Package ‘Seurat’

October 17, 2021

Version 4.0.5
Date 2021-10-04
Title Tools for Single Cell Genomics
Description A toolkit for quality control, analysis, and exploration of single cell RNA sequenc-
ing data. 'Seurat' aims to enable users to identify and interpret sources of heterogeneity from sin-
gle cell transcriptomic measurements, and to integrate diverse types of sin-
gle cell data. See Satija R, Farrell J, Gennert D, et al (2015) <doi:10.1038/nbt.3192>, Ma-
cosko E, Basu A, Satija R, et al (2015) <doi:10.1016/j.cell.2015.05.002>, Stuart T, But-
ler A, et al (2019) <doi:10.1016/j.cell.2019.05.031>, and Hao, Hao, et al (2020) <doi:10.1101/2020.10.12.335331> for more
tails.
URL https://satijalab.org/seurat, https://github.com/satijalab/seurat
BugReports https://github.com/satijalab/seurat/issues
Depends R (>= 4.0.0), methods
Imports cluster, cowplot, fitdistrplus, future, future.apply, ggplot2
(>= 3.3.0), ggrepel, ggridges, graphics, grDevices, grid, httr,
ica, igraph, irlba, jsonlite, KernSmooth, leiden (>= 0.3.1),
lmtest, MASS, Matrix (>= 1.2-14), matrixStats, miniUI,
patchwork, pbapply, plotly (>= 4.9.0), png, RANN, RColorBrewer,
Rcpp (>= 1.0.7), RcppAnnoy (>= 0.0.18), reticulate, rlang,
ROCR, Rtsne, scales, scattermore (>= 0.7), sctransform (>=
0.3.2), SeuratObject (>= 4.0.2), shiny, spatstat.core,
spatstat.geom, stats, tibble, tools, utils, uwot (>= 0.1.9)
LinkingTo Rcpp (>= 0.11.0), RcppEigen, RcppProgress
License GPL-3 | file LICENSE
LazyData true
Collate 'RcppExports.R' 'reexports.R' 'generics.R' 'clustering.R'
'visualization.R' 'convenience.R' 'data.R'
'differential_expression.R' 'dimensional_reduction.R'
'integration.R' 'mixscape.R' 'objects.R' 'preprocessing.R'
'tree.R' 'utilities.R' 'zzz.R'
RoxygenNote 7.1.1
Encoding UTF-8

1
2 R topics documented:

Suggests ape, rsvd, testthat, hdf5r, S4Vectors, SummarizedExperiment,

SingleCellExperiment, MAST, DESeq2, BiocGenerics,
GenomicRanges, GenomeInfoDb, IRanges, rtracklayer, Rfast2,
monocle, Biobase, VGAM, limma, metap, enrichR, mixtools
NeedsCompilation yes
Author Andrew Butler [ctb] (<https://orcid.org/0000-0003-3608-0463>),
Saket Choudhary [ctb] (<https://orcid.org/0000-0001-5202-7633>),
Charlotte Darby [ctb] (<https://orcid.org/0000-0003-2195-5300>),
Jeff Farrell [ctb],
Christoph Hafemeister [ctb] (<https://orcid.org/0000-0001-6365-8254>),
Yuhan Hao [ctb] (<https://orcid.org/0000-0002-1810-0822>),
Paul Hoffman [aut, cre] (<https://orcid.org/0000-0002-7693-8957>),
Jaison Jain [ctb] (<https://orcid.org/0000-0002-9478-5018>),
Efthymia Papalexi [ctb] (<https://orcid.org/0000-0001-5898-694X>),
Patrick Roelli [ctb],
Rahul Satija [ctb] (<https://orcid.org/0000-0001-9448-8833>),
Karthik Shekhar [ctb],
Avi Srivastava [ctb] (<https://orcid.org/0000-0001-9798-2079>),
Tim Stuart [ctb] (<https://orcid.org/0000-0002-3044-0897>),
Kristof Torkenczy [ctb] (<https://orcid.org/0000-0002-4869-7957>),
Shiwei Zheng [ctb] (<https://orcid.org/0000-0001-6682-6743>),
Satija Lab and Collaborators [fnd]
Maintainer Paul Hoffman <[email protected]>
Repository CRAN
Date/Publication 2021-10-17 20:30:02 UTC

R topics documented:
Seurat-package . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
AddAzimuthResults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
AddAzimuthScores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
AddModuleScore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
AggregateExpression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
AnchorSet-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
AnnotateAnchors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
as.CellDataSet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
as.Seurat.CellDataSet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
as.SingleCellExperiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
as.sparse.H5Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Assay-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
AugmentPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
AverageExpression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
BarcodeInflectionsPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
BGTextColor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
BlackAndWhite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
BuildClusterTree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
R topics documented: 3

CalcPerturbSig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
CalculateBarcodeInflections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
CaseMatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
cc.genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
cc.genes.updated.2019 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
CellCycleScoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Cells.SCTModel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
CellsByImage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
CellScatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
CellSelector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
CollapseEmbeddingOutliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
CollapseSpeciesExpressionMatrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
ColorDimSplit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
CombinePlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
contrast-theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
CreateSCTAssayObject . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
CustomDistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
DEenrichRPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
DietSeurat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
DimHeatmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
DimPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
DimReduc-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
DiscretePalette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
DoHeatmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
DotPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
ElbowPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
ExpMean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
ExpSD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
ExpVar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
FastRowScale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
FeaturePlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
FeatureScatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
FilterSlideSeq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
FindAllMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
FindClusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
FindConservedMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
FindIntegrationAnchors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
FindMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
FindMultiModalNeighbors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
FindNeighbors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
FindSpatiallyVariableFeatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
FindSubCluster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
FindTransferAnchors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
FindVariableFeatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
FoldChange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
GetAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
GetImage.SlideSeq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
GetIntegrationData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4 R topics documented:

GetResidual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
GetTissueCoordinates.SlideSeq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
GetTransferPredictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Graph-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
GroupCorrelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
GroupCorrelationPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
HoverLocator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
HTODemux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
HTOHeatmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
HVFInfo.SCTAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
IFeaturePlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
IntegrateData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
IntegrateEmbeddings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
IntegrationAnchorSet-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
IntegrationData-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
ISpatialDimPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
ISpatialFeaturePlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
JackStraw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
JackStrawData-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
JackStrawPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
L2CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
L2Dim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
LabelClusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
LabelPoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
LinkedPlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Load10X_Spatial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
LoadAnnoyIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
LoadSTARmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
LocalStruct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
LogNormalize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
LogVMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
MappingScore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
MapQuery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
merge.SCTAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
MetaFeature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
MinMax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
MixingMetric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
MixscapeHeatmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
MixscapeLDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
ModalityWeights-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
MULTIseqDemux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Neighbor-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
NNPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
NormalizeData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
PCASigGenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
PercentageFeatureSet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
PlotClusterTree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
PlotPerturbScore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
R topics documented: 5

PolyDimPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
PolyFeaturePlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
PredictAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
PrepLDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
PrepSCTIntegration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
ProjectDim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
ProjectUMAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Radius.SlideSeq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Read10X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Read10X_h5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Read10X_Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
ReadMtx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
ReadParseBio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
ReadSlideSeq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
ReadSTARsolo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
RegroupIdents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
RelativeCounts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
RenameCells.SCTAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
RidgePlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
RunCCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
RunICA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
RunLDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
RunMarkVario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
RunMixscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
RunMoransI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
RunPCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
RunSPCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
RunTSNE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
RunUMAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
SampleUMI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
SaveAnnoyIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
ScaleData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
ScaleFactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
ScoreJackStraw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
SCTAssay-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
SCTransform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
SCTResults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
SelectIntegrationFeatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
SetIntegrationData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Seurat-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
SeuratCommand-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
SeuratTheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
SlideSeq-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
SpatialImage-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
SpatialPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
SplitObject . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
STARmap-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
subset.AnchorSet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
6 Seurat-package

SubsetByBarcodeInflections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
TopCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
TopFeatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
TopNeighbors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
TransferAnchorSet-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
TransferData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
UpdateSCTAssays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
UpdateSymbolList . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
VariableFeaturePlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
VisiumV1-class . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
VizDimLoadings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
VlnPlot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Index 207

Seurat-package Seurat: Tools for Single Cell Genomics

Description
A toolkit for quality control, analysis, and exploration of single cell RNA sequencing data. ’Seu-
rat’ aims to enable users to identify and interpret sources of heterogeneity from single cell tran-
scriptomic measurements, and to integrate diverse types of single cell data. See Satija R, Farrell
J, Gennert D, et al (2015) <doi:10.1038/nbt.3192>, Macosko E, Basu A, Satija R, et al (2015)
<doi:10.1016/j.cell.2015.05.002>, Stuart T, Butler A, et al (2019) <doi:10.1016/j.cell.2019.05.031>,
and Hao, Hao, et al (2020) <doi:10.1101/2020.10.12.335331> for more details.

Package options
Seurat uses the following [options()] to configure behaviour:

Seurat.memsafe global option to call gc() after many operations. This can be helpful in cleaning
up the memory status of the R session and prevent use of swap space. However, it does add to
the computational overhead and setting to FALSE can speed things up if you’re working in an
environment where RAM availability is not a concern.
Seurat.warn.umap.uwot Show warning about the default backend for RunUMAP changing from
Python UMAP via reticulate to UWOT
Seurat.checkdots For functions that have ... as a parameter, this controls the behavior when an
item isn’t used. Can be one of warn, stop, or silent.
Seurat.limma.wilcox.msg Show message about more efficient Wilcoxon Rank Sum test avail-
able via the limma package
Seurat.Rfast2.msg Show message about more efficient Moran’s I function available via the
Rfast2 package
Seurat.warn.vlnplot.split Show message about changes to default behavior of split/multi vi-
olin plots
AddAzimuthResults 7

Author(s)
Maintainer: Paul Hoffman <[email protected]> (ORCID)
Other contributors:

• Andrew Butler <[email protected]> (ORCID) [contributor]

• Saket Choudhary <[email protected]> (ORCID) [contributor]
• Charlotte Darby <[email protected]> (ORCID) [contributor]
• Jeff Farrell <[email protected]> [contributor]
• Christoph Hafemeister <[email protected]> (ORCID) [contributor]
• Yuhan Hao <[email protected]> (ORCID) [contributor]
• Jaison Jain <[email protected]> (ORCID) [contributor]
• Efthymia Papalexi <[email protected]> (ORCID) [contributor]
• Patrick Roelli <[email protected]> [contributor]
• Rahul Satija <[email protected]> (ORCID) [contributor]
• Karthik Shekhar <[email protected]> [contributor]
• Avi Srivastava <[email protected]> (ORCID) [contributor]
• Tim Stuart <[email protected]> (ORCID) [contributor]
• Kristof Torkenczy (ORCID) [contributor]
• Shiwei Zheng <[email protected]> (ORCID) [contributor]
• Satija Lab and Collaborators [funder]

See Also
Useful links:

• https://satijalab.org/seurat
• https://github.com/satijalab/seurat
• Report bugs at https://github.com/satijalab/seurat/issues

AddAzimuthResults Add Azimuth Results

Description
Add mapping and prediction scores, UMAP embeddings, and imputed assay (if available) from
Azimuth to an existing or new Seurat object

Usage
AddAzimuthResults(object = NULL, filename)
8 AddAzimuthScores

Arguments
object A Seurat object
filename Path to Azimuth mapping scores file

Value
object with Azimuth results added

Examples
## Not run:
object <- AddAzimuthResults(object, filename = "azimuth_results.Rds")

## End(Not run)

AddAzimuthScores Add Azimuth Scores

Description
Add mapping and prediction scores from Azimuth to a Seurat object

Usage
AddAzimuthScores(object, filename)

Arguments
object A Seurat object
filename Path to Azimuth mapping scores file

Value
object with the mapping scores added

Examples
## Not run:
object <- AddAzimuthScores(object, filename = "azimuth_pred.tsv")

## End(Not run)
AddModuleScore 9

AddModuleScore Calculate module scores for feature expression programs in single

cells

Description
Calculate the average expression levels of each program (cluster) on single cell level, subtracted
by the aggregated expression of control feature sets. All analyzed features are binned based on
averaged expression, and the control features are randomly selected from each bin.

Usage
AddModuleScore(
object,
features,
pool = NULL,
nbin = 24,
ctrl = 100,
k = FALSE,
assay = NULL,
name = "Cluster",
seed = 1,
search = FALSE,
...
)

Arguments
object Seurat object
features A list of vectors of features for expression programs; each entry should be a
vector of feature names
pool List of features to check expression levels against, defaults to rownames(x =
object)
nbin Number of bins of aggregate expression levels for all analyzed features
ctrl Number of control features selected from the same bin per analyzed feature
k Use feature clusters returned from DoKMeans
assay Name of assay to use
name Name for the expression programs; will append a number to the end for each
entry in features (eg. if features has three programs, the results will be
stored as name1, name2, name3, respectively)
seed Set a random seed. If NULL, seed is not set.
search Search for symbol synonyms for features in features that don’t match features
in object? Searches the HGNC’s gene names database; see UpdateSymbolList
for more details
... Extra parameters passed to UpdateSymbolList
10 AggregateExpression

Value

Returns a Seurat object with module scores added to object meta data; each module is stored as
name# for each module program present in features

References

Tirosh et al, Science (2016)

Examples
## Not run:
data("pbmc_small")
cd_features <- list(c(
'CD79B',
'CD79A',
'CD19',
'CD180',
'CD200',
'CD3D',
'CD2',
'CD3E',
'CD7',
'CD8A',
'CD14',
'CD1C',
'CD68',
'CD9',
'CD247'
))
pbmc_small <- AddModuleScore(
object = pbmc_small,
features = cd_features,
ctrl = 5,
name = 'CD_Features'
)
head(x = pbmc_small[])

## End(Not run)

AggregateExpression Aggregated feature expression by identity class

Description

Returns aggregated (summed) expression values for each identity class

AggregateExpression 11

Usage

AggregateExpression(
object,
assays = NULL,
features = NULL,
return.seurat = FALSE,
group.by = "ident",
add.ident = NULL,
slot = "data",
verbose = TRUE,
...
)

Arguments

object Seurat object

assays Which assays to use. Default is all assays
features Features to analyze. Default is all features in the assay
return.seurat Whether to return the data as a Seurat object. Default is FALSE
group.by Categories for grouping (e.g, ident, replicate, celltype); ’ident’ by default
add.ident (Deprecated) Place an additional label on each cell prior to pseudobulking (very
useful if you want to observe cluster pseudobulk values, separated by replicate,
for example)
slot Slot(s) to use; if multiple slots are given, assumed to follow the order of ’assays’
(if specified) or object’s assays
verbose Print messages and show progress bar
... Arguments to be passed to methods such as CreateSeuratObject#’

Details

If slot is set to ’data’, this function assumes that the data has been log normalized and therefore fea-
ture values are exponentiated prior to aggregating so that sum is done in non-log space. Otherwise,
if slot is set to either ’counts’ or ’scale.data’, no exponentiation is performed prior to aggregating
If return.seurat = TRUE and slot is not ’scale.data’, aggregated values are placed in the ’counts’
slot of the returned object and the log of aggregated values are placed in the ’data’ slot. For the
ScaleData is then run on the default assay before returning the object. If return.seurat = TRUE
and slot is ’scale.data’, the ’counts’ slot is left empty, the ’data’ slot is filled with NA, and ’scale.data’
is set to the aggregated values.

Value

Returns a matrix with genes as rows, identity classes as columns. If return.seurat is TRUE, returns
an object of class Seurat.
12 AnnotateAnchors

Examples

data("pbmc_small")
head(AggregateExpression(object = pbmc_small))

AnchorSet-class The AnchorSet Class

Description

The AnchorSet class is an intermediate data storage class that stores the anchors and other related in-
formation needed for performing downstream analyses - namely data integration (IntegrateData)
and data transfer (TransferData).

Slots

object.list List of objects used to create anchors

reference.cells List of cell names in the reference dataset - needed when performing data trans-
fer.
reference.objects Position of reference object/s in object.list
query.cells List of cell names in the query dataset - needed when performing data transfer
anchors The anchor matrix. This contains the cell indices of both anchor pair cells, the anchor
score, and the index of the original dataset in the object.list for cell1 and cell2 of the anchor.
offsets The offsets used to enable cell look up in downstream functions
anchor.features The features used when performing anchor finding.
neighbors List containing Neighbor objects for reuse later (e.g. mapping)
command Store log of parameters that were used

AnnotateAnchors Add info to anchor matrix

Description

Add info to anchor matrix

AnnotateAnchors 13

Usage

AnnotateAnchors(anchors, vars, slot, ...)

## Default S3 method:
AnnotateAnchors(
anchors,
vars = NULL,
slot = NULL,
object.list,
assay = NULL,
...
)

## S3 method for class 'IntegrationAnchorSet'

AnnotateAnchors(
anchors,
vars = NULL,
slot = NULL,
object.list = NULL,
assay = NULL,
...
)

## S3 method for class 'TransferAnchorSet'

AnnotateAnchors(
anchors,
vars = NULL,
slot = NULL,
reference = NULL,
query = NULL,
assay = NULL,
...
)

Arguments

anchors An AnchorSet object

vars Variables to pull for each object via FetchData
slot Slot to pull feature data for
... Arguments passed to other methods
object.list List of Seurat objects
assay Specify the Assay per object if annotating with expression data
reference Reference object used in FindTransferAnchors
query Query object used in FindTransferAnchors
14 as.Seurat.CellDataSet

Value
Returns the anchor dataframe with additional columns for annotation metadata

as.CellDataSet Convert objects to CellDataSet objects

Description
Convert objects to CellDataSet objects

Usage
as.CellDataSet(x, ...)

## S3 method for class 'Seurat'

as.CellDataSet(x, assay = NULL, reduction = NULL, ...)

Arguments
x An object to convert to class CellDataSet
... Arguments passed to other methods
assay Assay to convert
reduction Name of DimReduc to set to main reducedDim in cds

as.Seurat.CellDataSet Convert objects to Seurat objects

Description
Convert objects to Seurat objects

Usage
## S3 method for class 'CellDataSet'
as.Seurat(x, slot = "counts", assay = "RNA", verbose = TRUE, ...)

## S3 method for class 'SingleCellExperiment'

as.Seurat(
x,
counts = "counts",
data = "logcounts",
assay = NULL,
project = "SingleCellExperiment",
...
)
as.SingleCellExperiment 15

Arguments
x An object to convert to class Seurat
slot Slot to store expression data as
assay Name of assays to convert; set to NULL for all assays to be converted
verbose Show progress updates
... Arguments passed to other methods
counts name of the SingleCellExperiment assay to store as counts; set to NULL if only
normalized data are present
data name of the SingleCellExperiment assay to slot as data. Set to NULL if only
counts are present
project Project name for new Seurat object

Value
A Seurat object generated from x

See Also
SeuratObject::as.Seurat

as.SingleCellExperiment
Convert objects to SingleCellExperiment objects

Description
Convert objects to SingleCellExperiment objects

Usage
as.SingleCellExperiment(x, ...)

## S3 method for class 'Seurat'

as.SingleCellExperiment(x, assay = NULL, ...)

Arguments
x An object to convert to class SingleCellExperiment
... Arguments passed to other methods
assay Assays to convert
16 as.sparse.H5Group

as.sparse.H5Group Cast to Sparse

Description
Cast to Sparse

Usage
## S3 method for class 'H5Group'
as.sparse(x, ...)

## S3 method for class 'Matrix'

as.data.frame(
x,
row.names = NULL,
optional = FALSE,
...,
stringsAsFactors = default.stringsAsFactors()
)

Arguments
x An object
... Arguments passed to other methods
row.names NULL or a character vector giving the row names for the data; missing values are
not allowed
optional logical. If TRUE, setting row names and converting column names (to syntac-
tic names: see make.names) is optional. Note that all of R’s base package
as.data.frame() methods use optional only for column names treatment,
basically with the meaning of data.frame(*,check.names = !optional). See
also the make.names argument of the matrix method.
stringsAsFactors
logical: should the character vector be converted to a factor?

Value
as.data.frame.Matrix: A data frame representation of the S4 Matrix

See Also
SeuratObject::as.sparse
Assay-class 17

Assay-class The Assay Class

Description
The Assay object is the basic unit of Seurat; for more details, please see the documentation in
SeuratObject

See Also
SeuratObject::Assay-class

AugmentPlot Augments ggplot2-based plot with a PNG image.

Description
Creates "vector-friendly" plots. Does this by saving a copy of the plot as a PNG file, then adding
the PNG image with annotation_raster to a blank plot of the same dimensions as plot. Please
note: original legends and axes will be lost during augmentation.

Usage
AugmentPlot(plot, width = 10, height = 10, dpi = 100)

Arguments
plot A ggplot object
width, height Width and height of PNG version of plot
dpi Plot resolution

Value
A ggplot object

Examples
## Not run:
data("pbmc_small")
plot <- DimPlot(object = pbmc_small)
AugmentPlot(plot = plot)

## End(Not run)
18 AverageExpression

AverageExpression Averaged feature expression by identity class

Description
Returns averaged expression values for each identity class

Usage
AverageExpression(
object,
assays = NULL,
features = NULL,
return.seurat = FALSE,
group.by = "ident",
add.ident = NULL,
slot = "data",
verbose = TRUE,
...
)

Arguments
object Seurat object
assays Which assays to use. Default is all assays
features Features to analyze. Default is all features in the assay
return.seurat Whether to return the data as a Seurat object. Default is FALSE
group.by Categories for grouping (e.g, ident, replicate, celltype); ’ident’ by default
add.ident (Deprecated) Place an additional label on each cell prior to pseudobulking (very
useful if you want to observe cluster pseudobulk values, separated by replicate,
for example)
slot Slot(s) to use; if multiple slots are given, assumed to follow the order of ’assays’
(if specified) or object’s assays
verbose Print messages and show progress bar
... Arguments to be passed to methods such as CreateSeuratObject

Details
If slot is set to ’data’, this function assumes that the data has been log normalized and therefore
feature values are exponentiated prior to averaging so that averaging is done in non-log space.
Otherwise, if slot is set to either ’counts’ or ’scale.data’, no exponentiation is performed prior to
averaging If return.seurat = TRUE and slot is not ’scale.data’, averaged values are placed in the
’counts’ slot of the returned object and the log of averaged values are placed in the ’data’ slot.
ScaleData is then run on the default assay before returning the object. If return.seurat = TRUE
and slot is ’scale.data’, the ’counts’ slot is left empty, the ’data’ slot is filled with NA, and ’scale.data’
is set to the aggregated values.
BarcodeInflectionsPlot 19

Value
Returns a matrix with genes as rows, identity classes as columns. If return.seurat is TRUE, returns
an object of class Seurat.

Examples
data("pbmc_small")
head(AverageExpression(object = pbmc_small))

BarcodeInflectionsPlot
Plot the Barcode Distribution and Calculated Inflection Points

Description
This function plots the calculated inflection points derived from the barcode-rank distribution.

Usage
BarcodeInflectionsPlot(object)

Arguments
object Seurat object

Details
See [CalculateBarcodeInflections()] to calculate inflection points and [SubsetByBarcodeInflections()]
to subsequently subset the Seurat object.

Value
Returns a ‘ggplot2‘ object showing the by-group inflection points and provided (or default) rank
threshold values in grey.

Author(s)
Robert A. Amezquita, <[email protected]>

See Also
CalculateBarcodeInflections SubsetByBarcodeInflections

Examples
data("pbmc_small")
pbmc_small <- CalculateBarcodeInflections(pbmc_small, group.column = 'groups')
BarcodeInflectionsPlot(pbmc_small)
20 BGTextColor

BGTextColor Determine text color based on background color

Description

Determine text color based on background color

Usage

BGTextColor(
background,
threshold = 186,
w3c = FALSE,
dark = "black",
light = "white"
)

Arguments

background A vector of background colors; supports R color names and hexadecimal codes
threshold Intensity threshold for light/dark cutoff; intensities greater than theshold yield
dark, others yield light
w3c Use W3C formula for calculating background text color; ignores threshold
dark Color for dark text
light Color for light text

Value

A named vector of either dark or light, depending on background; names of vector are background

Source

https://stackoverflow.com/questions/3942878/how-to-decide-font-color-in-white-or-black-depending-on

Examples

BGTextColor(background = c('black', 'white', '#E76BF3'))

BlackAndWhite 21

BlackAndWhite Create a custom color palette

Description

Creates a custom color palette based on low, middle, and high color values

Usage

BlackAndWhite(mid = NULL, k = 50)

BlueAndRed(k = 50)

CustomPalette(low = "white", high = "red", mid = NULL, k = 50)

PurpleAndYellow(k = 50)

Arguments

mid middle color. Optional.

k number of steps (colors levels) to include between low and high values
low low color
high high color

Value

A color palette for plotting

Examples
df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
plot(df, col = BlackAndWhite())

df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
plot(df, col = BlueAndRed())

myPalette <- CustomPalette()

myPalette

df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
plot(df, col = PurpleAndYellow())
22 BuildClusterTree

BuildClusterTree Phylogenetic Analysis of Identity Classes

Description
Constructs a phylogenetic tree relating the ’average’ cell from each identity class. Tree is estimated
based on a distance matrix constructed in either gene expression space or PCA space.

Usage
BuildClusterTree(
object,
assay = NULL,
features = NULL,
dims = NULL,
reduction = "pca",
graph = NULL,
slot = "data",
reorder = FALSE,
reorder.numeric = FALSE,
verbose = TRUE
)

Arguments
object Seurat object
assay Assay to use for the analysis.
features Genes to use for the analysis. Default is the set of variable genes (VariableFeatures(object
= object))
dims If set, tree is calculated in dimension reduction space; overrides features
reduction Name of dimension reduction to use. Only used if dims is not NULL.
graph If graph is passed, build tree based on graph connectivity between clusters; over-
rides dims and features
slot Slot(s) to use; if multiple slots are given, assumed to follow the order of ’assays’
(if specified) or object’s assays
reorder Re-order identity classes (factor ordering), according to position on the tree.
This groups similar classes together which can be helpful, for example, when
drawing violin plots.
reorder.numeric
Re-order identity classes according to position on the tree, assigning a numeric
value (’1’ is the leftmost node)
verbose Show progress updates
CalcPerturbSig 23

Details
Note that the tree is calculated for an ’average’ cell, so gene expression or PC scores are averaged
across all cells in an identity class before the tree is constructed.

Value
A Seurat object where the cluster tree can be accessed with Tool

Examples
if (requireNamespace("ape", quietly = TRUE)) {
data("pbmc_small")
pbmc_small
pbmc_small <- BuildClusterTree(object = pbmc_small)
Tool(object = pbmc_small, slot = 'BuildClusterTree')
}

CalcPerturbSig Calculate a perturbation Signature

Description
Function to calculate perturbation signature for pooled CRISPR screen datasets. For each target cell
(expressing one target gRNA), we identified 20 cells from the control pool (non-targeting cells) with
the most similar mRNA expression profiles. The perturbation signature is calculated by subtracting
the averaged mRNA expression profile of the non-targeting neighbors from the mRNA expression
profile of the target cell.

Usage
CalcPerturbSig(
object,
assay = NULL,
features = NULL,
slot = "data",
gd.class = "guide_ID",
nt.cell.class = "NT",
split.by = NULL,
num.neighbors = NULL,
reduction = "pca",
ndims = 15,
new.assay.name = "PRTB",
verbose = TRUE
)
24 CalculateBarcodeInflections

Arguments

object An object of class Seurat.

assay Name of Assay PRTB signature is being calculated on.
features Features to compute PRTB signature for. Defaults to the variable features set in
the assay specified.
slot Data slot to use for PRTB signature calculation.
gd.class Metadata column containing target gene classification.
nt.cell.class Non-targeting gRNA cell classification identity.
split.by Provide metadata column if multiple biological replicates exist to calculate PRTB
signature for every replicate separately.
num.neighbors Number of nearest neighbors to consider.
reduction Reduction method used to calculate nearest neighbors.
ndims Number of dimensions to use from dimensionality reduction method.
new.assay.name Name for the new assay.
verbose Display progress + messages

Value

Returns a Seurat object with a new assay added containing the perturbation signature for all cells in
the data slot.

CalculateBarcodeInflections
Calculate the Barcode Distribution Inflection

Description

This function calculates an adaptive inflection point ("knee") of the barcode distribution for each
sample group. This is useful for determining a threshold for removing low-quality samples.

Usage

CalculateBarcodeInflections(
object,
barcode.column = "nCount_RNA",
group.column = "orig.ident",
threshold.low = NULL,
threshold.high = NULL
)
CalculateBarcodeInflections 25

Arguments

object Seurat object

barcode.column Column to use as proxy for barcodes ("nCount_RNA" by default)
group.column Column to group by ("orig.ident" by default)
threshold.low Ignore barcodes of rank below this threshold in inflection calculation
threshold.high Ignore barcodes of rank above thisf threshold in inflection calculation

Details

The function operates by calculating the slope of the barcode number vs. rank distribution, and
then finding the point at which the distribution changes most steeply (the "knee"). Of note, this
calculation often must be restricted as to the range at which it performs, so ‘threshold‘ parameters
are provided to restrict the range of the calculation based on the rank of the barcodes. [BarcodeIn-
flectionsPlot()] is provided as a convenience function to visualize and test different thresholds and
thus provide more sensical end results.
See [BarcodeInflectionsPlot()] to visualize the calculated inflection points and [SubsetByBarcode-
Inflections()] to subsequently subset the Seurat object.

Value

Returns Seurat object with a new list in the ‘tools‘ slot, ‘CalculateBarcodeInflections‘ with values:
* ‘barcode_distribution‘ - contains the full barcode distribution across the entire dataset * ‘inflec-
tion_points‘ - the calculated inflection points within the thresholds * ‘threshold_values‘ - the pro-
vided (or default) threshold values to search within for inflections * ‘cells_pass‘ - the cells that pass
the inflection point calculation

Author(s)

Robert A. Amezquita, <[email protected]>

See Also

BarcodeInflectionsPlot SubsetByBarcodeInflections

Examples

data("pbmc_small")
CalculateBarcodeInflections(pbmc_small, group.column = 'groups')
26 cc.genes

CaseMatch Match the case of character vectors

Description
Match the case of character vectors

Usage
CaseMatch(search, match)

Arguments
search A vector of search terms
match A vector of characters whose case should be matched

Value
Values from search present in match with the case of match

Examples
data("pbmc_small")
cd_genes <- c('Cd79b', 'Cd19', 'Cd200')
CaseMatch(search = cd_genes, match = rownames(x = pbmc_small))

cc.genes Cell cycle genes

Description
A list of genes used in cell-cycle regression

Usage
cc.genes

Format
A list of two vectors
s.genes Genes associated with S-phase
g2m.genes Genes associated with G2M-phase

Source
https://www.science.org/doi/abs/10.1126/science.aad0501
cc.genes.updated.2019 27

cc.genes.updated.2019 Cell cycle genes: 2019 update

Description
A list of genes used in cell-cycle regression, updated with 2019 symbols

Usage
cc.genes.updated.2019

Format
A list of two vectors

s.genes Genes associated with S-phase

g2m.genes Genes associated with G2M-phase

Updated symbols
The following symbols were updated from cc.genes

s.genes • MCM2: MCM7

• MLF1IP: CENPU
• RPA2: POLR1B
• BRIP1: MRPL36
g2m.genes • FAM64A: PIMREG
• HN1: JPT1

Source
https://www.science.org/doi/abs/10.1126/science.aad0501

See Also
cc.genes

Examples
## Not run:
cc.genes.updated.2019 <- cc.genes
cc.genes.updated.2019$s.genes <- UpdateSymbolList(symbols = cc.genes.updated.2019$s.genes)
cc.genes.updated.2019$g2m.genes <- UpdateSymbolList(symbols = cc.genes.updated.2019$g2m.genes)

## End(Not run)
28 CellCycleScoring

CellCycleScoring Score cell cycle phases

Description
Score cell cycle phases

Usage
CellCycleScoring(
object,
s.features,
g2m.features,
ctrl = NULL,
set.ident = FALSE,
...
)

Arguments
object A Seurat object
s.features A vector of features associated with S phase
g2m.features A vector of features associated with G2M phase
ctrl Number of control features selected from the same bin per analyzed feature
supplied to AddModuleScore. Defaults to value equivalent to minimum number
of features present in ’s.features’ and ’g2m.features’.
set.ident If true, sets identity to phase assignments Stashes old identities in ’old.ident’
... Arguments to be passed to AddModuleScore

Value
A Seurat object with the following columns added to object meta data: S.Score, G2M.Score, and
Phase

See Also
AddModuleScore

Examples
## Not run:
data("pbmc_small")
# pbmc_small doesn't have any cell-cycle genes
# To run CellCycleScoring, please use a dataset with cell-cycle genes
# An example is available at http://satijalab.org/seurat/cell_cycle_vignette.html
pbmc_small <- CellCycleScoring(
object = pbmc_small,
Cells.SCTModel 29

g2m.features = cc.genes$g2m.genes,
s.features = cc.genes$s.genes
)
head(x = [email protected])

## End(Not run)

Cells.SCTModel Get Cell Names

Description
Get Cell Names

Usage
## S3 method for class 'SCTModel'
Cells(x)

## S3 method for class 'SlideSeq'

Cells(x)

## S3 method for class 'STARmap'

Cells(x)

## S3 method for class 'VisiumV1'

Cells(x)

Arguments
x An object

See Also
SeuratObject::Cells

CellsByImage Get a vector of cell names associated with an image (or set of images)

Description
Get a vector of cell names associated with an image (or set of images)

Usage
CellsByImage(object, images = NULL, unlist = FALSE)
30 CellScatter

Arguments
object Seurat object
images Vector of image names
unlist Return as a single vector of cell names as opposed to a list, named by image
name.

Value
A vector of cell names

Examples
## Not run:
CellsByImage(object = object, images = "slice1")

## End(Not run)

CellScatter Cell-cell scatter plot

Description
Creates a plot of scatter plot of features across two single cells. Pearson correlation between the
two cells is displayed above the plot.

Usage
CellScatter(
object,
cell1,
cell2,
features = NULL,
highlight = NULL,
cols = NULL,
pt.size = 1,
smooth = FALSE,
raster = NULL
)

Arguments
object Seurat object
cell1 Cell 1 name
cell2 Cell 2 name
features Features to plot (default, all features)
CellSelector 31

highlight Features to highlight

cols Colors to use for identity class plotting.
pt.size Size of the points on the plot
smooth Smooth the graph (similar to smoothScatter)
raster Convert points to raster format, default is NULL which will automatically use
raster if the number of points plotted is greater than 100,000

Value
A ggplot object

Examples
data("pbmc_small")
CellScatter(object = pbmc_small, cell1 = 'ATAGGAGAAACAGA', cell2 = 'CATCAGGATGCACA')

CellSelector Cell Selector

Description
Select points on a scatterplot and get information about them

Usage
CellSelector(plot, object = NULL, ident = "SelectedCells", ...)

FeatureLocator(plot, ...)

Arguments
plot A ggplot2 plot
object An optional Seurat object; if passes, will return an object with the identities of
selected cells set to ident
ident An optional new identity class to assign the selected cells
... Ignored

Value
If object is NULL, the names of the points selected; otherwise, a Seurat object with the selected
cells identity classes set to ident

See Also
DimPlot FeaturePlot
32 CollapseEmbeddingOutliers

Examples
## Not run:
data("pbmc_small")
plot <- DimPlot(object = pbmc_small)
# Follow instructions in the terminal to select points
cells.located <- CellSelector(plot = plot)
cells.located
# Automatically set the identity class of selected cells and return a new Seurat object
pbmc_small <- CellSelector(plot = plot, object = pbmc_small, ident = 'SelectedCells')

## End(Not run)

CollapseEmbeddingOutliers
Move outliers towards center on dimension reduction plot

Description
Move outliers towards center on dimension reduction plot

Usage
CollapseEmbeddingOutliers(
object,
reduction = "umap",
dims = 1:2,
group.by = "ident",
outlier.sd = 2,
reduction.key = "UMAP_"
)

Arguments
object Seurat object
reduction Name of DimReduc to adjust
dims Dimensions to visualize
group.by Group (color) cells in different ways (for example, orig.ident)
outlier.sd Controls the outlier distance
reduction.key Key for DimReduc that is returned

Value
Returns a DimReduc object with the modified embeddings
CollapseSpeciesExpressionMatrix 33

Examples
## Not run:
data("pbmc_small")
pbmc_small <- FindClusters(pbmc_small, resolution = 1.1)
pbmc_small <- RunUMAP(pbmc_small, dims = 1:5)
DimPlot(pbmc_small, reduction = "umap")
pbmc_small[["umap_new"]] <- CollapseEmbeddingOutliers(pbmc_small,
reduction = "umap", reduction.key = 'umap_', outlier.sd = 0.5)
DimPlot(pbmc_small, reduction = "umap_new")

## End(Not run)

CollapseSpeciesExpressionMatrix
Slim down a multi-species expression matrix, when only one species is
primarily of interenst.

Description

Valuable for CITE-seq analyses, where we typically spike in rare populations of ’negative control’
cells from a different species.

Usage

CollapseSpeciesExpressionMatrix(
object,
prefix = "HUMAN_",
controls = "MOUSE_",
ncontrols = 100
)

Arguments

object A UMI count matrix. Should contain rownames that start with the ensuing ar-
guments prefix.1 or prefix.2
prefix The prefix denoting rownames for the species of interest. Default is "HU-
MAN_". These rownames will have this prefix removed in the returned matrix.
controls The prefix denoting rownames for the species of ’negative control’ cells. Default
is "MOUSE_".
ncontrols How many of the most highly expressed (average) negative control features (by
default, 100 mouse genes), should be kept? All other rownames starting with
prefix.2 are discarded.
34 ColorDimSplit

Value
A UMI count matrix. Rownames that started with prefix have this prefix discarded. For rownames
starting with controls, only the ncontrols most highly expressed features are kept, and the prefix
is kept. All other rows are retained.

Examples
## Not run:
cbmc.rna.collapsed <- CollapseSpeciesExpressionMatrix(cbmc.rna)

## End(Not run)

ColorDimSplit Color dimensional reduction plot by tree split

Description
Returns a DimPlot colored based on whether the cells fall in clusters to the left or to the right of a
node split in the cluster tree.

Usage
ColorDimSplit(
object,
node,
left.color = "red",
right.color = "blue",
other.color = "grey50",
...
)

Arguments
object Seurat object
node Node in cluster tree on which to base the split
left.color Color for the left side of the split
right.color Color for the right side of the split
other.color Color for all other cells
... Arguments passed on to DimPlot
dims Dimensions to plot, must be a two-length numeric vector specifying x-
and y-dimensions
cells Vector of cells to plot (default is all cells)
ColorDimSplit 35

cols Vector of colors, each color corresponds to an identity class. This may
also be a single character or numeric value corresponding to a palette as
specified by brewer.pal.info. By default, ggplot2 assigns colors. We
also include a number of palettes from the pals package. See DiscretePalette
for details.
pt.size Adjust point size for plotting
reduction Which dimensionality reduction to use. If not specified, first searches
for umap, then tsne, then pca
group.by Name of one or more metadata columns to group (color) cells by (for
example, orig.ident); pass ’ident’ to group by identity class
split.by Name of a metadata column to split plot by; see FetchData for more
details
shape.by If NULL, all points are circles (default). You can specify any cell
attribute (that can be pulled with FetchData) allowing for both different
colors and different shapes on cells. Only applicable if raster = FALSE.
order Specify the order of plotting for the idents. This can be useful for crowded
plots if points of interest are being buried. Provide either a full list of valid
idents or a subset to be plotted last (on top)
shuffle Whether to randomly shuffle the order of points. This can be useful
for crowded plots if points of interest are being buried. (default is FALSE)
seed Sets the seed if randomly shuffling the order of points.
label Whether to label the clusters
label.size Sets size of labels
label.color Sets the color of the label text
label.box Whether to put a box around the label text (geom_text vs geom_label)
repel Repel labels
cells.highlight A list of character or numeric vectors of cells to highlight. If
only one group of cells desired, can simply pass a vector instead of a list. If
set, colors selected cells to the color(s) in cols.highlight and other cells
black (white if dark.theme = TRUE); will also resize to the size(s) passed
to sizes.highlight
cols.highlight A vector of colors to highlight the cells as; will repeat to the
length groups in cells.highlight
sizes.highlight Size of highlighted cells; will repeat to the length groups in
cells.highlight
na.value Color value for NA points when using custom scale
ncol Number of columns for display when combining plots
combine Combine plots into a single patchworked ggplot object. If FALSE,
return a list of ggplot objects
raster Convert points to raster format, default is NULL which automatically
rasterizes if plotting more than 100,000 cells

Value
Returns a DimPlot
36 CombinePlots

See Also
DimPlot

Examples
if (requireNamespace("ape", quietly = TRUE)) {
data("pbmc_small")
pbmc_small <- BuildClusterTree(object = pbmc_small, verbose = FALSE)
PlotClusterTree(pbmc_small)
ColorDimSplit(pbmc_small, node = 5)
}

CombinePlots Combine ggplot2-based plots into a single plot

Description
Combine ggplot2-based plots into a single plot

Usage
CombinePlots(plots, ncol = NULL, legend = NULL, ...)

Arguments
plots A list of gg objects
ncol Number of columns
legend Combine legends into a single legend choose from ’right’ or ’bottom’; pass
’none’ to remove legends, or NULL to leave legends as they are
... Extra parameters passed to plot_grid

Value
A combined plot

Examples
data("pbmc_small")
pbmc_small[['group']] <- sample(
x = c('g1', 'g2'),
size = ncol(x = pbmc_small),
replace = TRUE
)
plot1 <- FeaturePlot(
object = pbmc_small,
features = 'MS4A1',
split.by = 'group'
contrast-theory 37

)
plot2 <- FeaturePlot(
object = pbmc_small,
features = 'FCN1',
split.by = 'group'
)
CombinePlots(
plots = list(plot1, plot2),
legend = 'none',
nrow = length(x = unique(x = pbmc_small[['group', drop = TRUE]]))
)

contrast-theory Get the intensity and/or luminance of a color

Description
Get the intensity and/or luminance of a color

Usage
Intensity(color)

Luminance(color)

Arguments
color A vector of colors

Value
A vector of intensities/luminances for each color

Source
https://stackoverflow.com/questions/3942878/how-to-decide-font-color-in-white-or-black-depending-on

Examples
Intensity(color = c('black', 'white', '#E76BF3'))

Luminance(color = c('black', 'white', '#E76BF3'))

38 CreateSCTAssayObject

CreateSCTAssayObject Create a SCT Assay object

Description

Create a SCT object from a feature (e.g. gene) expression matrix and a list of SCTModels. The
expected format of the input matrix is features x cells.

Usage

CreateSCTAssayObject(
counts,
data,
scale.data = NULL,
umi.assay = "RNA",
min.cells = 0,
min.features = 0,
SCTModel.list = NULL
)

Arguments

counts Unnormalized data such as raw counts or TPMs

data Prenormalized data; if provided, do not pass counts
scale.data a residual matrix
umi.assay The UMI assay name. Default is RNA
min.cells Include features detected in at least this many cells. Will subset the counts
matrix as well. To reintroduce excluded features, create a new object with a
lower cutoff.
min.features Include cells where at least this many features are detected.
SCTModel.list list of SCTModels

Details

Non-unique cell or feature names are not allowed. Please make unique before calling this function.
CustomDistance 39

CustomDistance Run a custom distance function on an input data matrix

Description

Run a custom distance function on an input data matrix

Usage

CustomDistance(my.mat, my.function, ...)

Arguments

my.mat A matrix to calculate distance on

my.function A function to calculate distance
... Extra parameters to my.function

Value

A distance matrix

Author(s)

Jean Fan

Examples
data("pbmc_small")
# Define custom distance matrix
manhattan.distance <- function(x, y) return(sum(abs(x-y)))

input.data <- GetAssayData(pbmc_small, assay.type = "RNA", slot = "scale.data")

cell.manhattan.dist <- CustomDistance(input.data, manhattan.distance)

DEenrichRPlot DE and EnrichR pathway visualization barplot

Description

DE and EnrichR pathway visualization barplot

40 DEenrichRPlot

Usage
DEenrichRPlot(
object,
ident.1 = NULL,
ident.2 = NULL,
balanced = TRUE,
logfc.threshold = 0.25,
assay = NULL,
max.genes,
test.use = "wilcox",
p.val.cutoff = 0.05,
cols = NULL,
enrich.database = NULL,
num.pathway = 10,
return.gene.list = FALSE,
...
)

Arguments
object Name of object class Seurat.
ident.1 Cell class identity 1.
ident.2 Cell class identity 2.
balanced Option to display pathway enrichments for both negative and positive DE genes.If
false, only positive DE gene will be displayed.
logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.
assay Assay to use in differential expression testing
max.genes Maximum number of genes to use as input to enrichR.
test.use Denotes which test to use. Available options are:
• "wilcox" : Identifies differentially expressed genes between two groups of
cells using a Wilcoxon Rank Sum test (default)
• "bimod" : Likelihood-ratio test for single cell gene expression, (McDavid
et al., Bioinformatics, 2013)
• "roc" : Identifies ’markers’ of gene expression using ROC analysis. For
each gene, evaluates (using AUC) a classifier built on that gene alone, to
classify between two groups of cells. An AUC value of 1 means that ex-
pression values for this gene alone can perfectly classify the two groupings
(i.e. Each of the cells in cells.1 exhibit a higher level than each of the cells
in cells.2). An AUC value of 0 also means there is perfect classification, but
in the other direction. A value of 0.5 implies that the gene has no predictive
power to classify the two groups. Returns a ’predictive power’ (abs(AUC-
0.5) * 2) ranked matrix of putative differentially expressed genes.
DietSeurat 41

• "t" : Identify differentially expressed genes between two groups of cells

using the Student’s t-test.
• "negbinom" : Identifies differentially expressed genes between two groups
of cells using a negative binomial generalized linear model. Use only for
UMI-based datasets
• "poisson" : Identifies differentially expressed genes between two groups of
cells using a poisson generalized linear model. Use only for UMI-based
datasets
• "LR" : Uses a logistic regression framework to determine differentially
expressed genes. Constructs a logistic regression model predicting group
membership based on each feature individually and compares this to a null
model with a likelihood ratio test.
• "MAST" : Identifies differentially expressed genes between two groups of
cells using a hurdle model tailored to scRNA-seq data. Utilizes the MAST
package to run the DE testing.
• "DESeq2" : Identifies differentially expressed genes between two groups of
cells based on a model using DESeq2 which uses a negative binomial distri-
bution (Love et al, Genome Biology, 2014).This test does not support pre-
filtering of genes based on average difference (or percent detection rate) be-
tween cell groups. However, genes may be pre-filtered based on their min-
imum detection rate (min.pct) across both cell groups. To use this method,
please install DESeq2, using the instructions at https://bioconductor.org/packages/release/bioc/html/D
p.val.cutoff Cutoff to select DE genes.
cols A list of colors to use for barplots.
enrich.database
Database to use from enrichR.
num.pathway Number of pathways to display in barplot.
return.gene.list
Return list of DE genes
... Arguments passed to other methods and to specific DE methods

Value
Returns one (only enriched) or two (both enriched and depleted) barplots with the top enriched/depleted
GO terms from EnrichR.

DietSeurat Slim down a Seurat object

Description
Keep only certain aspects of the Seurat object. Can be useful in functions that utilize merge as it
reduces the amount of data in the merge.
42 DimHeatmap

Usage
DietSeurat(
object,
counts = TRUE,
data = TRUE,
scale.data = FALSE,
features = NULL,
assays = NULL,
dimreducs = NULL,
graphs = NULL
)

Arguments
object Seurat object
counts Preserve the count matrices for the assays specified
data Preserve the data slot for the assays specified
scale.data Preserve the scale.data slot for the assays specified
features Only keep a subset of features, defaults to all features
assays Only keep a subset of assays specified here
dimreducs Only keep a subset of DimReducs specified here (if NULL, remove all DimRe-
ducs)
graphs Only keep a subset of Graphs specified here (if NULL, remove all Graphs)

DimHeatmap Dimensional reduction heatmap

Description
Draws a heatmap focusing on a principal component. Both cells and genes are sorted by their
principal component scores. Allows for nice visualization of sources of heterogeneity in the dataset.

Usage
DimHeatmap(
object,
dims = 1,
nfeatures = 30,
cells = NULL,
reduction = "pca",
disp.min = -2.5,
disp.max = NULL,
balanced = TRUE,
projected = FALSE,
DimHeatmap 43

ncol = NULL,
fast = TRUE,
raster = TRUE,
slot = "scale.data",
assays = NULL,
combine = TRUE
)

PCHeatmap(object, ...)

Arguments
object Seurat object
dims Dimensions to plot
nfeatures Number of genes to plot
cells A list of cells to plot. If numeric, just plots the top cells.
reduction Which dimmensional reduction to use
disp.min Minimum display value (all values below are clipped)
disp.max Maximum display value (all values above are clipped); defaults to 2.5 if slot is
’scale.data’, 6 otherwise
balanced Plot an equal number of genes with both + and - scores.
projected Use the full projected dimensional reduction
ncol Number of columns to plot
fast If true, use image to generate plots; faster than using ggplot2, but not customiz-
able
raster If true, plot with geom_raster, else use geom_tile. geom_raster may look blurry
on some viewing applications such as Preview due to how the raster is interpo-
lated. Set this to FALSE if you are encountering that issue (note that plots may
take longer to produce/render).
slot Data slot to use, choose from ’raw.data’, ’data’, or ’scale.data’
assays A vector of assays to pull data from
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot objects
... Extra parameters passed to DimHeatmap

Value
No return value by default. If using fast = FALSE, will return a patchworked ggplot object if
combine = TRUE, otherwise returns a list of ggplot objects

See Also
image geom_raster
44 DimPlot

Examples
data("pbmc_small")
DimHeatmap(object = pbmc_small)

DimPlot Dimensional reduction plot

Description
Graphs the output of a dimensional reduction technique on a 2D scatter plot where each point is a
cell and it’s positioned based on the cell embeddings determined by the reduction technique. By
default, cells are colored by their identity class (can be changed with the group.by parameter).

Usage
DimPlot(
object,
dims = c(1, 2),
cells = NULL,
cols = NULL,
pt.size = NULL,
reduction = NULL,
group.by = NULL,
split.by = NULL,
shape.by = NULL,
order = NULL,
shuffle = FALSE,
seed = 1,
label = FALSE,
label.size = 4,
label.color = "black",
label.box = FALSE,
repel = FALSE,
cells.highlight = NULL,
cols.highlight = "#DE2D26",
sizes.highlight = 1,
na.value = "grey50",
ncol = NULL,
combine = TRUE,
raster = NULL
)

PCAPlot(object, ...)

TSNEPlot(object, ...)

UMAPPlot(object, ...)
DimPlot 45

Arguments
object Seurat object
dims Dimensions to plot, must be a two-length numeric vector specifying x- and y-
dimensions
cells Vector of cells to plot (default is all cells)
cols Vector of colors, each color corresponds to an identity class. This may also
be a single character or numeric value corresponding to a palette as specified
by brewer.pal.info. By default, ggplot2 assigns colors. We also include a
number of palettes from the pals package. See DiscretePalette for details.
pt.size Adjust point size for plotting
reduction Which dimensionality reduction to use. If not specified, first searches for umap,
then tsne, then pca
group.by Name of one or more metadata columns to group (color) cells by (for example,
orig.ident); pass ’ident’ to group by identity class
split.by Name of a metadata column to split plot by; see FetchData for more details
shape.by If NULL, all points are circles (default). You can specify any cell attribute (that
can be pulled with FetchData) allowing for both different colors and different
shapes on cells. Only applicable if raster = FALSE.
order Specify the order of plotting for the idents. This can be useful for crowded plots
if points of interest are being buried. Provide either a full list of valid idents or a
subset to be plotted last (on top)
shuffle Whether to randomly shuffle the order of points. This can be useful for crowded
plots if points of interest are being buried. (default is FALSE)
seed Sets the seed if randomly shuffling the order of points.
label Whether to label the clusters
label.size Sets size of labels
label.color Sets the color of the label text
label.box Whether to put a box around the label text (geom_text vs geom_label)
repel Repel labels
cells.highlight
A list of character or numeric vectors of cells to highlight. If only one group
of cells desired, can simply pass a vector instead of a list. If set, colors se-
lected cells to the color(s) in cols.highlight and other cells black (white if
dark.theme = TRUE); will also resize to the size(s) passed to sizes.highlight
cols.highlight A vector of colors to highlight the cells as; will repeat to the length groups in
cells.highlight
sizes.highlight
Size of highlighted cells; will repeat to the length groups in cells.highlight
na.value Color value for NA points when using custom scale
ncol Number of columns for display when combining plots
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot objects
46 DimReduc-class

raster Convert points to raster format, default is NULL which automatically rasterizes if
plotting more than 100,000 cells
... Extra parameters passed to DimPlot

Value

A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

Note

For the old do.hover and do.identify functionality, please see HoverLocator and CellSelector,
respectively.

See Also

FeaturePlot HoverLocator CellSelector FetchData

Examples

data("pbmc_small")
DimPlot(object = pbmc_small)
DimPlot(object = pbmc_small, split.by = 'ident')

DimReduc-class The DimReduc Class

Description

The DimReduc object stores a dimensionality reduction taken out in Seurat; for more details, please
see the documentation in SeuratObject

See Also

SeuratObject::DimReduc-class
DiscretePalette 47

DiscretePalette Discrete colour palettes from the pals package

Description
These are included here because pals depends on a number of compiled packages, and this can lead
to increases in run time for Travis, and generally should be avoided when possible.

Usage
DiscretePalette(n, palette = NULL)

Arguments
n Number of colours to be generated.
palette Options are "alphabet", "alphabet2", "glasbey", "polychrome", and "stepped".
Can be omitted and the function will use the one based on the requested n.

Details
These palettes are a much better default for data with many classes than the default ggplot2 palette.
Many thanks to Kevin Wright for writing the pals package.
Taken from the pals package (Licence: GPL-3). https://cran.r-project.org/package=pals
Credit: Kevin Wright

Value
A vector of colors

DoHeatmap Feature expression heatmap

Description
Draws a heatmap of single cell feature expression.

Usage
DoHeatmap(
object,
features = NULL,
cells = NULL,
group.by = "ident",
group.bar = TRUE,
group.colors = NULL,
48 DoHeatmap

disp.min = -2.5,
disp.max = NULL,
slot = "scale.data",
assay = NULL,
label = TRUE,
size = 5.5,
hjust = 0,
angle = 45,
raster = TRUE,
draw.lines = TRUE,
lines.width = NULL,
group.bar.height = 0.02,
combine = TRUE
)

Arguments
object Seurat object
features A vector of features to plot, defaults to VariableFeatures(object = object)
cells A vector of cells to plot
group.by A vector of variables to group cells by; pass ’ident’ to group by cell identity
classes
group.bar Add a color bar showing group status for cells
group.colors Colors to use for the color bar
disp.min Minimum display value (all values below are clipped)
disp.max Maximum display value (all values above are clipped); defaults to 2.5 if slot is
’scale.data’, 6 otherwise
slot Data slot to use, choose from ’raw.data’, ’data’, or ’scale.data’
assay Assay to pull from
label Label the cell identies above the color bar
size Size of text above color bar
hjust Horizontal justification of text above color bar
angle Angle of text above color bar
raster If true, plot with geom_raster, else use geom_tile. geom_raster may look blurry
on some viewing applications such as Preview due to how the raster is interpo-
lated. Set this to FALSE if you are encountering that issue (note that plots may
take longer to produce/render).
draw.lines Include white lines to separate the groups
lines.width Integer number to adjust the width of the separating white lines. Corresponds to
the number of "cells" between each group.
group.bar.height
Scale the height of the color bar
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot objects
DotPlot 49

Value
A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

Examples
data("pbmc_small")
DoHeatmap(object = pbmc_small)

DotPlot Dot plot visualization

Description
Intuitive way of visualizing how feature expression changes across different identity classes (clus-
ters). The size of the dot encodes the percentage of cells within a class, while the color encodes the
AverageExpression level across all cells within a class (blue is high).

Usage
DotPlot(
object,
assay = NULL,
features,
cols = c("lightgrey", "blue"),
col.min = -2.5,
col.max = 2.5,
dot.min = 0,
dot.scale = 6,
idents = NULL,
group.by = NULL,
split.by = NULL,
cluster.idents = FALSE,
scale = TRUE,
scale.by = "radius",
scale.min = NA,
scale.max = NA
)

Arguments
object Seurat object
assay Name of assay to use, defaults to the active assay
features Input vector of features, or named list of feature vectors if feature-grouped pan-
els are desired (replicates the functionality of the old SplitDotPlotGG)
50 DotPlot

cols Colors to plot: the name of a palette from RColorBrewer::brewer.pal.info,

a pair of colors defining a gradient, or 3+ colors defining multiple gradients (if
split.by is set)
col.min Minimum scaled average expression threshold (everything smaller will be set to
this)
col.max Maximum scaled average expression threshold (everything larger will be set to
this)
dot.min The fraction of cells at which to draw the smallest dot (default is 0). All cell
groups with less than this expressing the given gene will have no dot drawn.
dot.scale Scale the size of the points, similar to cex
idents Identity classes to include in plot (default is all)
group.by Factor to group the cells by
split.by Factor to split the groups by (replicates the functionality of the old SplitDot-
PlotGG); see FetchData for more details
cluster.idents Whether to order identities by hierarchical clusters based on given features, de-
fault is FALSE
scale Determine whether the data is scaled, TRUE for default
scale.by Scale the size of the points by ’size’ or by ’radius’
scale.min Set lower limit for scaling, use NA for default
scale.max Set upper limit for scaling, use NA for default

Value

A ggplot object

See Also

RColorBrewer::brewer.pal.info

Examples

data("pbmc_small")
cd_genes <- c("CD247", "CD3E", "CD9")
DotPlot(object = pbmc_small, features = cd_genes)
pbmc_small[['groups']] <- sample(x = c('g1', 'g2'), size = ncol(x = pbmc_small), replace = TRUE)
DotPlot(object = pbmc_small, features = cd_genes, split.by = 'groups')
ElbowPlot 51

ElbowPlot Quickly Pick Relevant Dimensions

Description
Plots the standard deviations (or approximate singular values if running PCAFast) of the principle
components for easy identification of an elbow in the graph. This elbow often corresponds well
with the significant dims and is much faster to run than Jackstraw

Usage
ElbowPlot(object, ndims = 20, reduction = "pca")

Arguments
object Seurat object
ndims Number of dimensions to plot standard deviation for
reduction Reduction technique to plot standard deviation for

Value
A ggplot object

Examples
data("pbmc_small")
ElbowPlot(object = pbmc_small)

ExpMean Calculate the mean of logged values

Description
Calculate mean of logged values in non-log space (return answer in log-space)

Usage
ExpMean(x, ...)

Arguments
x A vector of values
... Other arguments (not used)
52 ExpVar

Value
Returns the mean in log-space

Examples
ExpMean(x = c(1, 2, 3))

ExpSD Calculate the standard deviation of logged values

Description
Calculate SD of logged values in non-log space (return answer in log-space)

Usage
ExpSD(x)

Arguments
x A vector of values

Value
Returns the standard deviation in log-space

Examples
ExpSD(x = c(1, 2, 3))

ExpVar Calculate the variance of logged values

Description
Calculate variance of logged values in non-log space (return answer in log-space)

Usage
ExpVar(x)

Arguments
x A vector of values
FastRowScale 53

Value

Returns the variance in log-space

Examples
ExpVar(x = c(1, 2, 3))

FastRowScale Scale and/or center matrix rowwise

Description

Performs row scaling and/or centering. Equivalent to using t(scale(t(mat))) in R except in the case
of NA values.

Usage

FastRowScale(mat, center = TRUE, scale = TRUE, scale_max = 10)

Arguments

mat A matrix
center a logical value indicating whether to center the rows
scale a logical value indicating whether to scale the rows
scale_max clip all values greater than scale_max to scale_max. Don’t clip if Inf.

Value

Returns the center/scaled matrix

FeaturePlot Visualize ’features’ on a dimensional reduction plot

Description

Colors single cells on a dimensional reduction plot according to a ’feature’ (i.e. gene expression,
PC scores, number of genes detected, etc.)
54 FeaturePlot

Usage
FeaturePlot(
object,
features,
dims = c(1, 2),
cells = NULL,
cols = if (blend) { c("lightgrey", "#ff0000", "#00ff00") } else {
c("lightgrey", "blue") },
pt.size = NULL,
order = FALSE,
min.cutoff = NA,
max.cutoff = NA,
reduction = NULL,
split.by = NULL,
keep.scale = "feature",
shape.by = NULL,
slot = "data",
blend = FALSE,
blend.threshold = 0.5,
label = FALSE,
label.size = 4,
repel = FALSE,
ncol = NULL,
coord.fixed = FALSE,
by.col = TRUE,
sort.cell = NULL,
interactive = FALSE,
combine = TRUE,
raster = NULL
)

Arguments
object Seurat object
features Vector of features to plot. Features can come from:
• An Assay feature (e.g. a gene name - "MS4A1")
• A column name from meta.data (e.g. mitochondrial percentage - "per-
cent.mito")
• A column name from a DimReduc object corresponding to the cell embed-
ding values (e.g. the PC 1 scores - "PC_1")
dims Dimensions to plot, must be a two-length numeric vector specifying x- and y-
dimensions
cells Vector of cells to plot (default is all cells)
cols The two colors to form the gradient over. Provide as string vector with the first
color corresponding to low values, the second to high. Also accepts a Brewer
color scale or vector of colors. Note: this will bin the data into number of colors
provided. When blend is TRUE, takes anywhere from 1-3 colors:
FeaturePlot 55

1 color: Treated as color for double-negatives, will use default colors 2 and 3
for per-feature expression
2 colors: Treated as colors for per-feature expression, will use default color 1
for double-negatives
3+ colors: First color used for double-negatives, colors 2 and 3 used for per-
feature expression, all others ignored
pt.size Adjust point size for plotting
order Boolean determining whether to plot cells in order of expression. Can be useful
if cells expressing given feature are getting buried.
min.cutoff, max.cutoff
Vector of minimum and maximum cutoff values for each feature, may specify
quantile in the form of ’q##’ where ’##’ is the quantile (eg, ’q1’, ’q10’)
reduction Which dimensionality reduction to use. If not specified, first searches for umap,
then tsne, then pca
split.by A factor in object metadata to split the feature plot by, pass ’ident’ to split by
cell identity’; similar to the old FeatureHeatmap
keep.scale How to handle the color scale across multiple plots. Options are:
• "feature" (default; by row/feature scaling): The plots for each individual
feature are scaled to the maximum expression of the feature across the con-
ditions provided to ’split.by’.
• "all" (universal scaling): The plots for all features and conditions are scaled
to the maximum expression value for the feature with the highest overall
expression.
• NULL (no scaling): Each individual plot is scaled to the maximum expres-
sion value of the feature in the condition provided to ’split.by’. Be aware
setting NULL will result in color scales that are not comparable between
plots.
shape.by If NULL, all points are circles (default). You can specify any cell attribute (that
can be pulled with FetchData) allowing for both different colors and different
shapes on cells. Only applicable if raster = FALSE.
slot Which slot to pull expression data from?
blend Scale and blend expression values to visualize coexpression of two features
blend.threshold
The color cutoff from weak signal to strong signal; ranges from 0 to 1.
label Whether to label the clusters
label.size Sets size of labels
repel Repel labels
ncol Number of columns to combine multiple feature plots to, ignored if split.by
is not NULL
coord.fixed Plot cartesian coordinates with fixed aspect ratio
by.col If splitting by a factor, plot the splits per column with the features as rows;
ignored if blend = TRUE
56 FeatureScatter

sort.cell Redundant with order. This argument is being deprecated. Please use order
instead.
interactive Launch an interactive FeaturePlot
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot objects
raster Convert points to raster format, default is NULL which automatically rasterizes if
plotting more than 100,000 cells

Value
A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

Note
For the old do.hover and do.identify functionality, please see HoverLocator and CellSelector,
respectively.

See Also
DimPlot HoverLocator CellSelector

Examples
data("pbmc_small")
FeaturePlot(object = pbmc_small, features = 'PC_1')

FeatureScatter Scatter plot of single cell data

Description
Creates a scatter plot of two features (typically feature expression), across a set of single cells. Cells
are colored by their identity class. Pearson correlation between the two features is displayed above
the plot.

Usage
FeatureScatter(
object,
feature1,
feature2,
cells = NULL,
shuffle = FALSE,
seed = 1,
group.by = NULL,
cols = NULL,
FeatureScatter 57

pt.size = 1,
shape.by = NULL,
span = NULL,
smooth = FALSE,
combine = TRUE,
slot = "data",
plot.cor = TRUE,
raster = NULL,
jitter = TRUE
)

Arguments
object Seurat object
feature1 First feature to plot. Typically feature expression but can also be metrics, PC
scores, etc. - anything that can be retreived with FetchData
feature2 Second feature to plot.
cells Cells to include on the scatter plot.
shuffle Whether to randomly shuffle the order of points. This can be useful for crowded
plots if points of interest are being buried. (default is FALSE)
seed Sets the seed if randomly shuffling the order of points.
group.by Name of one or more metadata columns to group (color) cells by (for example,
orig.ident); pass ’ident’ to group by identity class
cols Colors to use for identity class plotting.
pt.size Size of the points on the plot
shape.by Ignored for now
span Spline span in loess function call, if NULL, no spline added
smooth Smooth the graph (similar to smoothScatter)
combine Combine plots into a single patchworked
slot Slot to pull data from, should be one of ’counts’, ’data’, or ’scale.data’
plot.cor Display correlation in plot title
raster Convert points to raster format, default is NULL which will automatically use
raster if the number of points plotted is greater than 100,000
jitter Jitter for easier visualization of crowded points

Value
A ggplot object

Examples
data("pbmc_small")
FeatureScatter(object = pbmc_small, feature1 = 'CD9', feature2 = 'CD3E')
58 FilterSlideSeq

FilterSlideSeq Filter stray beads from Slide-seq puck

Description
This function is useful for removing stray beads that fall outside the main Slide-seq puck area.
Essentially, it’s a circular filter where you set a center and radius defining a circle of beads to keep.
If the center is not set, it will be estimated from the bead coordinates (removing the 1st and 99th
quantile to avoid skewing the center by the stray beads). By default, this function will display
a SpatialDimPlot showing which cells were removed for easy adjustment of the center and/or
radius.

Usage
FilterSlideSeq(
object,
image = "image",
center = NULL,
radius = NULL,
do.plot = TRUE
)

Arguments
object Seurat object with slide-seq data
image Name of the image where the coordinates are stored
center Vector specifying the x and y coordinates for the center of the inclusion circle
radius Radius of the circle of inclusion
do.plot Display a SpatialDimPlot with the cells being removed labeled.

Value
Returns a Seurat object with only the subset of cells that pass the circular filter

Examples
## Not run:
# This example uses the ssHippo dataset which you can download
# using the SeuratData package.
library(SeuratData)
data('ssHippo')
# perform filtering of beads
ssHippo.filtered <- FilterSlideSeq(ssHippo, radius = 2300)
# This radius looks to small so increase and repeat until satisfied

## End(Not run)
FindAllMarkers 59

FindAllMarkers Gene expression markers for all identity classes

Description
Finds markers (differentially expressed genes) for each of the identity classes in a dataset

Usage
FindAllMarkers(
object,
assay = NULL,
features = NULL,
logfc.threshold = 0.25,
test.use = "wilcox",
slot = "data",
min.pct = 0.1,
min.diff.pct = -Inf,
node = NULL,
verbose = TRUE,
only.pos = FALSE,
max.cells.per.ident = Inf,
random.seed = 1,
latent.vars = NULL,
min.cells.feature = 3,
min.cells.group = 3,
pseudocount.use = 1,
mean.fxn = NULL,
fc.name = NULL,
base = 2,
return.thresh = 0.01,
densify = FALSE,
...
)

Arguments
object An object
assay Assay to use in differential expression testing
features Genes to test. Default is to use all genes
logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.
test.use Denotes which test to use. Available options are:
60 FindAllMarkers

• "wilcox" : Identifies differentially expressed genes between two groups of

cells using a Wilcoxon Rank Sum test (default)
• "bimod" : Likelihood-ratio test for single cell gene expression, (McDavid
et al., Bioinformatics, 2013)
• "roc" : Identifies ’markers’ of gene expression using ROC analysis. For
each gene, evaluates (using AUC) a classifier built on that gene alone, to
classify between two groups of cells. An AUC value of 1 means that ex-
pression values for this gene alone can perfectly classify the two groupings
(i.e. Each of the cells in cells.1 exhibit a higher level than each of the cells
in cells.2). An AUC value of 0 also means there is perfect classification, but
in the other direction. A value of 0.5 implies that the gene has no predictive
power to classify the two groups. Returns a ’predictive power’ (abs(AUC-
0.5) * 2) ranked matrix of putative differentially expressed genes.
• "t" : Identify differentially expressed genes between two groups of cells
using the Student’s t-test.
• "negbinom" : Identifies differentially expressed genes between two groups
of cells using a negative binomial generalized linear model. Use only for
UMI-based datasets
• "poisson" : Identifies differentially expressed genes between two groups of
cells using a poisson generalized linear model. Use only for UMI-based
datasets
• "LR" : Uses a logistic regression framework to determine differentially
expressed genes. Constructs a logistic regression model predicting group
membership based on each feature individually and compares this to a null
model with a likelihood ratio test.
• "MAST" : Identifies differentially expressed genes between two groups of
cells using a hurdle model tailored to scRNA-seq data. Utilizes the MAST
package to run the DE testing.
• "DESeq2" : Identifies differentially expressed genes between two groups of
cells based on a model using DESeq2 which uses a negative binomial distri-
bution (Love et al, Genome Biology, 2014).This test does not support pre-
filtering of genes based on average difference (or percent detection rate) be-
tween cell groups. However, genes may be pre-filtered based on their min-
imum detection rate (min.pct) across both cell groups. To use this method,
please install DESeq2, using the instructions at https://bioconductor.org/packages/release/bioc/html/D
slot Slot to pull data from; note that if test.use is "negbinom", "poisson", or "DE-
Seq2", slot will be set to "counts"
min.pct only test genes that are detected in a minimum fraction of min.pct cells in either
of the two populations. Meant to speed up the function by not testing genes that
are very infrequently expressed. Default is 0.1
min.diff.pct only test genes that show a minimum difference in the fraction of detection
between the two groups. Set to -Inf by default
node A node to find markers for and all its children; requires BuildClusterTree to
have been run previously; replaces FindAllMarkersNode
verbose Print a progress bar once expression testing begins
only.pos Only return positive markers (FALSE by default)
FindAllMarkers 61

max.cells.per.ident
Down sample each identity class to a max number. Default is no downsampling.
Not activated by default (set to Inf)
random.seed Random seed for downsampling
latent.vars Variables to test, used only when test.use is one of ’LR’, ’negbinom’, ’pois-
son’, or ’MAST’
min.cells.feature
Minimum number of cells expressing the feature in at least one of the two
groups, currently only used for poisson and negative binomial tests
min.cells.group
Minimum number of cells in one of the groups
pseudocount.use
Pseudocount to add to averaged expression values when calculating logFC. 1 by
default.
mean.fxn Function to use for fold change or average difference calculation. If NULL, the
appropriate function will be chose according to the slot used
fc.name Name of the fold change, average difference, or custom function column in the
output data.frame. If NULL, the fold change column will be named according to
the logarithm base (eg, "avg_log2FC"), or if using the scale.data slot "avg_diff".
base The base with respect to which logarithms are computed.
return.thresh Only return markers that have a p-value < return.thresh, or a power > return.thresh
(if the test is ROC)
densify Convert the sparse matrix to a dense form before running the DE test. This can
provide speedups but might require higher memory; default is FALSE
... Arguments passed to other methods and to specific DE methods

Value
Matrix containing a ranked list of putative markers, and associated statistics (p-values, ROC score,
etc.)

Examples
data("pbmc_small")
# Find markers for all clusters
all.markers <- FindAllMarkers(object = pbmc_small)
head(x = all.markers)
## Not run:
# Pass a value to node as a replacement for FindAllMarkersNode
pbmc_small <- BuildClusterTree(object = pbmc_small)
all.markers <- FindAllMarkers(object = pbmc_small, node = 4)
head(x = all.markers)

## End(Not run)
62 FindClusters

FindClusters Cluster Determination

Description
Identify clusters of cells by a shared nearest neighbor (SNN) modularity optimization based clus-
tering algorithm. First calculate k-nearest neighbors and construct the SNN graph. Then optimize
the modularity function to determine clusters. For a full description of the algorithms, see Walt-
man and van Eck (2013) The European Physical Journal B. Thanks to Nigel Delaney (evolvedmi-
crobe@github) for the rewrite of the Java modularity optimizer code in Rcpp!

Usage
FindClusters(object, ...)

## Default S3 method:
FindClusters(
object,
modularity.fxn = 1,
initial.membership = NULL,
node.sizes = NULL,
resolution = 0.8,
method = "matrix",
algorithm = 1,
n.start = 10,
n.iter = 10,
random.seed = 0,
group.singletons = TRUE,
temp.file.location = NULL,
edge.file.name = NULL,
verbose = TRUE,
...
)

## S3 method for class 'Seurat'

FindClusters(
object,
graph.name = NULL,
modularity.fxn = 1,
initial.membership = NULL,
node.sizes = NULL,
resolution = 0.8,
method = "matrix",
algorithm = 1,
n.start = 10,
n.iter = 10,
random.seed = 0,
FindClusters 63

group.singletons = TRUE,
temp.file.location = NULL,
edge.file.name = NULL,
verbose = TRUE,
...
)

Arguments
object An object
... Arguments passed to other methods
modularity.fxn Modularity function (1 = standard; 2 = alternative).
initial.membership, node.sizes
Parameters to pass to the Python leidenalg function.
resolution Value of the resolution parameter, use a value above (below) 1.0 if you want to
obtain a larger (smaller) number of communities.
method Method for running leiden (defaults to matrix which is fast for small datasets).
Enable method = "igraph" to avoid casting large data to a dense matrix.
algorithm Algorithm for modularity optimization (1 = original Louvain algorithm; 2 =
Louvain algorithm with multilevel refinement; 3 = SLM algorithm; 4 = Leiden
algorithm). Leiden requires the leidenalg python.
n.start Number of random starts.
n.iter Maximal number of iterations per random start.
random.seed Seed of the random number generator.
group.singletons
Group singletons into nearest cluster. If FALSE, assign all singletons to a "sin-
gleton" group
temp.file.location
Directory where intermediate files will be written. Specify the ABSOLUTE
path.
edge.file.name Edge file to use as input for modularity optimizer jar.
verbose Print output
graph.name Name of graph to use for the clustering algorithm

Details
To run Leiden algorithm, you must first install the leidenalg python package (e.g. via pip install
leidenalg), see Traag et al (2018).

Value
Returns a Seurat object where the idents have been updated with new cluster info; latest clustering
results will be stored in object metadata under ’seurat_clusters’. Note that ’seurat_clusters’ will be
overwritten everytime FindClusters is run
64 FindConservedMarkers

FindConservedMarkers Finds markers that are conserved between the groups

Description
Finds markers that are conserved between the groups

Usage
FindConservedMarkers(
object,
ident.1,
ident.2 = NULL,
grouping.var,
assay = "RNA",
slot = "data",
meta.method = metap::minimump,
verbose = TRUE,
...
)

Arguments
object An object
ident.1 Identity class to define markers for
ident.2 A second identity class for comparison. If NULL (default) - use all other cells
for comparison.
grouping.var grouping variable
assay of assay to fetch data for (default is RNA)
slot Slot to pull data from; note that if test.use is "negbinom", "poisson", or "DE-
Seq2", slot will be set to "counts"
meta.method method for combining p-values. Should be a function from the metap package
(NOTE: pass the function, not a string)
verbose Print a progress bar once expression testing begins
... parameters to pass to FindMarkers

Value
data.frame containing a ranked list of putative conserved markers, and associated statistics (p-values
within each group and a combined p-value (such as Fishers combined p-value or others from the
metap package), percentage of cells expressing the marker, average differences). Name of group is
appended to each associated output column (e.g. CTRL_p_val). If only one group is tested in the
grouping.var, max and combined p-values are not returned.
FindIntegrationAnchors 65

Examples
## Not run:
data("pbmc_small")
pbmc_small
# Create a simulated grouping variable
pbmc_small[['groups']] <- sample(x = c('g1', 'g2'), size = ncol(x = pbmc_small), replace = TRUE)
FindConservedMarkers(pbmc_small, ident.1 = 0, ident.2 = 1, grouping.var = "groups")

## End(Not run)

FindIntegrationAnchors
Find integration anchors

Description
Find a set of anchors between a list of Seurat objects. These anchors can later be used to integrate
the objects using the IntegrateData function.

Usage
FindIntegrationAnchors(
object.list = NULL,
assay = NULL,
reference = NULL,
anchor.features = 2000,
scale = TRUE,
normalization.method = c("LogNormalize", "SCT"),
sct.clip.range = NULL,
reduction = c("cca", "rpca", "rlsi"),
l2.norm = TRUE,
dims = 1:30,
k.anchor = 5,
k.filter = 200,
k.score = 30,
max.features = 200,
nn.method = "annoy",
n.trees = 50,
eps = 0,
verbose = TRUE
)

Arguments
object.list A list of Seurat objects between which to find anchors for downstream integra-
tion.
66 FindIntegrationAnchors

assay A vector of assay names specifying which assay to use when constructing an-
chors. If NULL, the current default assay for each object is used.
reference A vector specifying the object/s to be used as a reference during integration. If
NULL (default), all pairwise anchors are found (no reference/s). If not NULL,
the corresponding objects in object.list will be used as references. When us-
ing a set of specified references, anchors are first found between each query and
each reference. The references are then integrated through pairwise integration.
Each query is then mapped to the integrated reference.
anchor.features
Can be either:
• A numeric value. This will call SelectIntegrationFeatures to select the
provided number of features to be used in anchor finding
• A vector of features to be used as input to the anchor finding process
scale Whether or not to scale the features provided. Only set to FALSE if you have
previously scaled the features you want to use for each object in the object.list
normalization.method
Name of normalization method used: LogNormalize or SCT
sct.clip.range Numeric of length two specifying the min and max values the Pearson residual
will be clipped to
reduction Dimensional reduction to perform when finding anchors. Can be one of:
• cca: Canonical correlation analysis
• rpca: Reciprocal PCA
• rlsi: Reciprocal LSI
l2.norm Perform L2 normalization on the CCA cell embeddings after dimensional re-
duction
dims Which dimensions to use from the CCA to specify the neighbor search space
k.anchor How many neighbors (k) to use when picking anchors
k.filter How many neighbors (k) to use when filtering anchors
k.score How many neighbors (k) to use when scoring anchors
max.features The maximum number of features to use when specifying the neighborhood
search space in the anchor filtering
nn.method Method for nearest neighbor finding. Options include: rann, annoy
n.trees More trees gives higher precision when using annoy approximate nearest neigh-
bor search
eps Error bound on the neighbor finding algorithm (from RANN/Annoy)
verbose Print progress bars and output

Details
The main steps of this procedure are outlined below. For a more detailed description of the method-
ology, please see Stuart, Butler, et al Cell 2019: doi: 10.1016/j.cell.2019.05.031; doi: 10.1101/
460147
First, determine anchor.features if not explicitly specified using SelectIntegrationFeatures.
Then for all pairwise combinations of reference and query datasets:
FindIntegrationAnchors 67

• Perform dimensional reduction on the dataset pair as specified via the reduction parameter.
If l2.norm is set to TRUE, perform L2 normalization of the embedding vectors.
• Identify anchors - pairs of cells from each dataset that are contained within each other’s neigh-
borhoods (also known as mutual nearest neighbors).
• Filter low confidence anchors to ensure anchors in the low dimension space are in broad
agreement with the high dimensional measurements. This is done by looking at the neighbors
of each query cell in the reference dataset using max.features to define this space. If the
reference cell isn’t found within the first k.filter neighbors, remove the anchor.
• Assign each remaining anchor a score. For each anchor cell, determine the nearest k.score
anchors within its own dataset and within its pair’s dataset. Based on these neighborhoods,
construct an overall neighbor graph and then compute the shared neighbor overlap between
anchor and query cells (analogous to an SNN graph). We use the 0.01 and 0.90 quantiles on
these scores to dampen outlier effects and rescale to range between 0-1.

Value
Returns an AnchorSet object that can be used as input to IntegrateData.

References
Stuart T, Butler A, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019;177:1888-1902
doi: 10.1016/j.cell.2019.05.031

Examples
## Not run:
# to install the SeuratData package see https://github.com/satijalab/seurat-data
library(SeuratData)
data("panc8")

# panc8 is a merged Seurat object containing 8 separate pancreas datasets

# split the object by dataset
pancreas.list <- SplitObject(panc8, split.by = "tech")

# perform standard preprocessing on each object

for (i in 1:length(pancreas.list)) {
pancreas.list[[i]] <- NormalizeData(pancreas.list[[i]], verbose = FALSE)
pancreas.list[[i]] <- FindVariableFeatures(
pancreas.list[[i]], selection.method = "vst",
nfeatures = 2000, verbose = FALSE
)
}

# find anchors
anchors <- FindIntegrationAnchors(object.list = pancreas.list)

# integrate data
integrated <- IntegrateData(anchorset = anchors)

## End(Not run)
68 FindMarkers

FindMarkers Gene expression markers of identity classes

Description
Finds markers (differentially expressed genes) for identity classes

Usage
FindMarkers(object, ...)

## Default S3 method:
FindMarkers(
object,
slot = "data",
counts = numeric(),
cells.1 = NULL,
cells.2 = NULL,
features = NULL,
logfc.threshold = 0.25,
test.use = "wilcox",
min.pct = 0.1,
min.diff.pct = -Inf,
verbose = TRUE,
only.pos = FALSE,
max.cells.per.ident = Inf,
random.seed = 1,
latent.vars = NULL,
min.cells.feature = 3,
min.cells.group = 3,
pseudocount.use = 1,
fc.results = NULL,
densify = FALSE,
...
)

## S3 method for class 'Assay'

FindMarkers(
object,
slot = "data",
cells.1 = NULL,
cells.2 = NULL,
features = NULL,
logfc.threshold = 0.25,
test.use = "wilcox",
min.pct = 0.1,
min.diff.pct = -Inf,
FindMarkers 69

verbose = TRUE,
only.pos = FALSE,
max.cells.per.ident = Inf,
random.seed = 1,
latent.vars = NULL,
min.cells.feature = 3,
min.cells.group = 3,
pseudocount.use = 1,
mean.fxn = NULL,
fc.name = NULL,
base = 2,
densify = FALSE,
...
)

## S3 method for class 'DimReduc'

FindMarkers(
object,
cells.1 = NULL,
cells.2 = NULL,
features = NULL,
logfc.threshold = 0.25,
test.use = "wilcox",
min.pct = 0.1,
min.diff.pct = -Inf,
verbose = TRUE,
only.pos = FALSE,
max.cells.per.ident = Inf,
random.seed = 1,
latent.vars = NULL,
min.cells.feature = 3,
min.cells.group = 3,
pseudocount.use = 1,
mean.fxn = rowMeans,
fc.name = NULL,
densify = FALSE,
...
)

## S3 method for class 'Seurat'

FindMarkers(
object,
ident.1 = NULL,
ident.2 = NULL,
group.by = NULL,
subset.ident = NULL,
assay = NULL,
slot = "data",
70 FindMarkers

reduction = NULL,
features = NULL,
logfc.threshold = 0.25,
test.use = "wilcox",
min.pct = 0.1,
min.diff.pct = -Inf,
verbose = TRUE,
only.pos = FALSE,
max.cells.per.ident = Inf,
random.seed = 1,
latent.vars = NULL,
min.cells.feature = 3,
min.cells.group = 3,
pseudocount.use = 1,
mean.fxn = NULL,
fc.name = NULL,
base = 2,
densify = FALSE,
...
)

Arguments
object An object
... Arguments passed to other methods and to specific DE methods
slot Slot to pull data from; note that if test.use is "negbinom", "poisson", or "DE-
Seq2", slot will be set to "counts"
counts Count matrix if using scale.data for DE tests. This is used for computing pct.1
and pct.2 and for filtering features based on fraction expressing
cells.1 Vector of cell names belonging to group 1
cells.2 Vector of cell names belonging to group 2
features Genes to test. Default is to use all genes
logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.
test.use Denotes which test to use. Available options are:
• "wilcox" : Identifies differentially expressed genes between two groups of
cells using a Wilcoxon Rank Sum test (default)
• "bimod" : Likelihood-ratio test for single cell gene expression, (McDavid
et al., Bioinformatics, 2013)
• "roc" : Identifies ’markers’ of gene expression using ROC analysis. For
each gene, evaluates (using AUC) a classifier built on that gene alone, to
classify between two groups of cells. An AUC value of 1 means that ex-
pression values for this gene alone can perfectly classify the two groupings
(i.e. Each of the cells in cells.1 exhibit a higher level than each of the cells
FindMarkers 71

in cells.2). An AUC value of 0 also means there is perfect classification, but

in the other direction. A value of 0.5 implies that the gene has no predictive
power to classify the two groups. Returns a ’predictive power’ (abs(AUC-
0.5) * 2) ranked matrix of putative differentially expressed genes.
• "t" : Identify differentially expressed genes between two groups of cells
using the Student’s t-test.
• "negbinom" : Identifies differentially expressed genes between two groups
of cells using a negative binomial generalized linear model. Use only for
UMI-based datasets
• "poisson" : Identifies differentially expressed genes between two groups of
cells using a poisson generalized linear model. Use only for UMI-based
datasets
• "LR" : Uses a logistic regression framework to determine differentially
expressed genes. Constructs a logistic regression model predicting group
membership based on each feature individually and compares this to a null
model with a likelihood ratio test.
• "MAST" : Identifies differentially expressed genes between two groups of
cells using a hurdle model tailored to scRNA-seq data. Utilizes the MAST
package to run the DE testing.
• "DESeq2" : Identifies differentially expressed genes between two groups of
cells based on a model using DESeq2 which uses a negative binomial distri-
bution (Love et al, Genome Biology, 2014).This test does not support pre-
filtering of genes based on average difference (or percent detection rate) be-
tween cell groups. However, genes may be pre-filtered based on their min-
imum detection rate (min.pct) across both cell groups. To use this method,
please install DESeq2, using the instructions at https://bioconductor.org/packages/release/bioc/html/D
min.pct only test genes that are detected in a minimum fraction of min.pct cells in either
of the two populations. Meant to speed up the function by not testing genes that
are very infrequently expressed. Default is 0.1
min.diff.pct only test genes that show a minimum difference in the fraction of detection
between the two groups. Set to -Inf by default
verbose Print a progress bar once expression testing begins
only.pos Only return positive markers (FALSE by default)
max.cells.per.ident
Down sample each identity class to a max number. Default is no downsampling.
Not activated by default (set to Inf)
random.seed Random seed for downsampling
latent.vars Variables to test, used only when test.use is one of ’LR’, ’negbinom’, ’pois-
son’, or ’MAST’
min.cells.feature
Minimum number of cells expressing the feature in at least one of the two
groups, currently only used for poisson and negative binomial tests
min.cells.group
Minimum number of cells in one of the groups
72 FindMarkers

pseudocount.use
Pseudocount to add to averaged expression values when calculating logFC. 1 by
default.
fc.results data.frame from FoldChange
densify Convert the sparse matrix to a dense form before running the DE test. This can
provide speedups but might require higher memory; default is FALSE
mean.fxn Function to use for fold change or average difference calculation. If NULL, the
appropriate function will be chose according to the slot used
fc.name Name of the fold change, average difference, or custom function column in the
output data.frame. If NULL, the fold change column will be named according to
the logarithm base (eg, "avg_log2FC"), or if using the scale.data slot "avg_diff".
base The base with respect to which logarithms are computed.
ident.1 Identity class to define markers for; pass an object of class phylo or ’clus-
tertree’ to find markers for a node in a cluster tree; passing ’clustertree’ requires
BuildClusterTree to have been run
ident.2 A second identity class for comparison; if NULL, use all other cells for compari-
son; if an object of class phylo or ’clustertree’ is passed to ident.1, must pass
a node to find markers for
group.by Regroup cells into a different identity class prior to performing differential ex-
pression (see example)
subset.ident Subset a particular identity class prior to regrouping. Only relevant if group.by
is set (see example)
assay Assay to use in differential expression testing
reduction Reduction to use in differential expression testing - will test for DE on cell em-
beddings

Details
p-value adjustment is performed using bonferroni correction based on the total number of genes in
the dataset. Other correction methods are not recommended, as Seurat pre-filters genes using the
arguments above, reducing the number of tests performed. Lastly, as Aaron Lun has pointed out,
p-values should be interpreted cautiously, as the genes used for clustering are the same genes tested
for differential expression.

Value
data.frame with a ranked list of putative markers as rows, and associated statistics as columns (p-
values, ROC score, etc., depending on the test used (test.use)). The following columns are always
present:

• avg_logFC: log fold-chage of the average expression between the two groups. Positive values
indicate that the gene is more highly expressed in the first group
• pct.1: The percentage of cells where the gene is detected in the first group
• pct.2: The percentage of cells where the gene is detected in the second group
• p_val_adj: Adjusted p-value, based on bonferroni correction using all genes in the dataset
FindMultiModalNeighbors 73

References
McDavid A, Finak G, Chattopadyay PK, et al. Data exploration, quality control and testing in
single-cell qPCR-based gene expression experiments. Bioinformatics. 2013;29(4):461-467. doi:10.1093/bioinformatics/bts71
Trapnell C, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal
ordering of single cells. Nature Biotechnology volume 32, pages 381-386 (2014)
Andrew McDavid, Greg Finak and Masanao Yajima (2017). MAST: Model-based Analysis of
Single Cell Transcriptomics. R package version 1.2.1. https://github.com/RGLab/MAST/
Love MI, Huber W and Anders S (2014). "Moderated estimation of fold change and dispersion for
RNA-seq data with DESeq2." Genome Biology. https://bioconductor.org/packages/release/bioc/html/DESeq2.html

See Also
FoldChange

Examples
data("pbmc_small")
# Find markers for cluster 2
markers <- FindMarkers(object = pbmc_small, ident.1 = 2)
head(x = markers)

# Take all cells in cluster 2, and find markers that separate cells in the 'g1' group (metadata
# variable 'group')
markers <- FindMarkers(pbmc_small, ident.1 = "g1", group.by = 'groups', subset.ident = "2")
head(x = markers)

# Pass 'clustertree' or an object of class phylo to ident.1 and

# a node to ident.2 as a replacement for FindMarkersNode
if (requireNamespace("ape", quietly = TRUE)) {
pbmc_small <- BuildClusterTree(object = pbmc_small)
markers <- FindMarkers(object = pbmc_small, ident.1 = 'clustertree', ident.2 = 5)
head(x = markers)
}

FindMultiModalNeighbors
Construct weighted nearest neighbor graph

Description
This function will construct a weighted nearest neighbor (WNN) graph. For each cell, we identify
the nearest neighbors based on a weighted combination of two modalities. Takes as input two
dimensional reductions, one computed for each modality.Other parameters are listed for debugging,
but can be left as default values.
74 FindMultiModalNeighbors

Usage
FindMultiModalNeighbors(
object,
reduction.list,
dims.list,
k.nn = 20,
l2.norm = TRUE,
knn.graph.name = "wknn",
snn.graph.name = "wsnn",
weighted.nn.name = "weighted.nn",
modality.weight.name = NULL,
knn.range = 200,
prune.SNN = 1/15,
sd.scale = 1,
cross.contant.list = NULL,
smooth = FALSE,
return.intermediate = FALSE,
modality.weight = NULL,
verbose = TRUE
)

Arguments
object A Seurat object
reduction.list A list of two dimensional reductions, one for each of the modalities to be inte-
grated
dims.list A list containing the dimensions for each reduction to use
k.nn the number of multimodal neighbors to compute. 20 by default
l2.norm Perform L2 normalization on the cell embeddings after dimensional reduction.
TRUE by default.
knn.graph.name Multimodal knn graph name
snn.graph.name Multimodal snn graph name
weighted.nn.name
Multimodal neighbor object name
modality.weight.name
Variable name to store modality weight in object meta data
knn.range The number of approximate neighbors to compute
prune.SNN Cutoff not to discard edge in SNN graph
sd.scale The scaling factor for kernel width. 1 by default
cross.contant.list
Constant used to avoid divide-by-zero errors. 1e-4 by default
smooth Smoothing modality score across each individual modality neighbors. FALSE
by default
return.intermediate
Store intermediate results in misc
FindNeighbors 75

modality.weight
A ModalityWeights object generated by FindModalityWeights
verbose Print progress bars and output

Value
Seurat object containing a nearest-neighbor object, KNN graph, and SNN graph - each based on a
weighted combination of modalities.

FindNeighbors (Shared) Nearest-neighbor graph construction

Description
Computes the k.param nearest neighbors for a given dataset. Can also optionally (via compute.SNN),
construct a shared nearest neighbor graph by calculating the neighborhood overlap (Jaccard index)
between every cell and its k.param nearest neighbors.

Usage
FindNeighbors(object, ...)

## Default S3 method:
FindNeighbors(
object,
query = NULL,
distance.matrix = FALSE,
k.param = 20,
return.neighbor = FALSE,
compute.SNN = !return.neighbor,
prune.SNN = 1/15,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "euclidean",
nn.eps = 0,
verbose = TRUE,
force.recalc = FALSE,
l2.norm = FALSE,
cache.index = FALSE,
index = NULL,
...
)

## S3 method for class 'Assay'

FindNeighbors(
object,
features = NULL,
76 FindNeighbors

k.param = 20,
return.neighbor = FALSE,
compute.SNN = !return.neighbor,
prune.SNN = 1/15,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "euclidean",
nn.eps = 0,
verbose = TRUE,
force.recalc = FALSE,
l2.norm = FALSE,
cache.index = FALSE,
...
)

## S3 method for class 'dist'

FindNeighbors(
object,
k.param = 20,
return.neighbor = FALSE,
compute.SNN = !return.neighbor,
prune.SNN = 1/15,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "euclidean",
nn.eps = 0,
verbose = TRUE,
force.recalc = FALSE,
l2.norm = FALSE,
cache.index = FALSE,
...
)

## S3 method for class 'Seurat'

FindNeighbors(
object,
reduction = "pca",
dims = 1:10,
assay = NULL,
features = NULL,
k.param = 20,
return.neighbor = FALSE,
compute.SNN = !return.neighbor,
prune.SNN = 1/15,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "euclidean",
nn.eps = 0,
FindNeighbors 77

verbose = TRUE,
force.recalc = FALSE,
do.plot = FALSE,
graph.name = NULL,
l2.norm = FALSE,
cache.index = FALSE,
...
)

Arguments
object An object
... Arguments passed to other methods
query Matrix of data to query against object. If missing, defaults to object.
distance.matrix
Boolean value of whether the provided matrix is a distance matrix; note, for
objects of class dist, this parameter will be set automatically
k.param Defines k for the k-nearest neighbor algorithm
return.neighbor
Return result as Neighbor object. Not used with distance matrix input.
compute.SNN also compute the shared nearest neighbor graph
prune.SNN Sets the cutoff for acceptable Jaccard index when computing the neighborhood
overlap for the SNN construction. Any edges with values less than or equal
to this will be set to 0 and removed from the SNN graph. Essentially sets the
stringency of pruning (0 — no pruning, 1 — prune everything).
nn.method Method for nearest neighbor finding. Options include: rann, annoy
n.trees More trees gives higher precision when using annoy approximate nearest neigh-
bor search
annoy.metric Distance metric for annoy. Options include: euclidean, cosine, manhattan, and
hamming
nn.eps Error bound when performing nearest neighbor seach using RANN; default of
0.0 implies exact nearest neighbor search
verbose Whether or not to print output to the console
force.recalc Force recalculation of (S)NN.
l2.norm Take L2Norm of the data
cache.index Include cached index in returned Neighbor object (only relevant if return.neighbor
= TRUE)
index Precomputed index. Useful if querying new data against existing index to avoid
recomputing.
features Features to use as input for building the (S)NN; used only when dims is NULL
reduction Reduction to use as input for building the (S)NN
dims Dimensions of reduction to use as input
assay Assay to use in construction of (S)NN; used only when dims is NULL
78 FindSpatiallyVariableFeatures

do.plot Plot SNN graph on tSNE coordinates

graph.name Optional naming parameter for stored (S)NN graph (or Neighbor object, if re-
turn.neighbor = TRUE). Default is assay.name_(s)nn. To store both the neighbor
graph and the shared nearest neighbor (SNN) graph, you must supply a vector
containing two names to the graph.name parameter. The first element in the
vector will be used to store the nearest neighbor (NN) graph, and the second
element used to store the SNN graph. If only one name is supplied, only the NN
graph is stored.

Value
This function can either return a Neighbor object with the KNN information or a list of Graph
objects with the KNN and SNN depending on the settings of return.neighbor and compute.SNN.
When running on a Seurat object, this returns the Seurat object with the Graphs or Neighbor
objects stored in their respective slots. Names of the Graph or Neighbor object can be found with
Graphs or Neighbors.

Examples
data("pbmc_small")
pbmc_small
# Compute an SNN on the gene expression level
pbmc_small <- FindNeighbors(pbmc_small, features = VariableFeatures(object = pbmc_small))

# More commonly, we build the SNN on a dimensionally reduced form of the data
# such as the first 10 principle components.

pbmc_small <- FindNeighbors(pbmc_small, reduction = "pca", dims = 1:10)

FindSpatiallyVariableFeatures
Find spatially variable features

Description
Identify features whose variability in expression can be explained to some degree by spatial location.

Usage
FindSpatiallyVariableFeatures(object, ...)

## Default S3 method:
FindSpatiallyVariableFeatures(
object,
spatial.location,
selection.method = c("markvariogram", "moransi"),
r.metric = 5,
FindSpatiallyVariableFeatures 79

x.cuts = NULL,
y.cuts = NULL,
verbose = TRUE,
...
)

## S3 method for class 'Assay'

FindSpatiallyVariableFeatures(
object,
slot = "scale.data",
spatial.location,
selection.method = c("markvariogram", "moransi"),
features = NULL,
r.metric = 5,
x.cuts = NULL,
y.cuts = NULL,
nfeatures = nfeatures,
verbose = TRUE,
...
)

## S3 method for class 'Seurat'

FindSpatiallyVariableFeatures(
object,
assay = NULL,
slot = "scale.data",
features = NULL,
image = NULL,
selection.method = c("markvariogram", "moransi"),
r.metric = 5,
x.cuts = NULL,
y.cuts = NULL,
nfeatures = 2000,
verbose = TRUE,
...
)

Arguments
object A Seurat object, assay, or expression matrix
... Arguments passed to other methods
spatial.location
Coordinates for each cell/spot/bead
selection.method
Method for selecting spatially variable features.
• markvariogram: See RunMarkVario for details
• moransi: See RunMoransI for details.
r.metric r value at which to report the "trans" value of the mark variogram
80 FindSubCluster

x.cuts Number of divisions to make in the x direction, helps define the grid over which
binning is performed
y.cuts Number of divisions to make in the y direction, helps define the grid over which
binning is performed
verbose Print messages and progress
slot Slot in the Assay to pull data from
features If provided, only compute on given features. Otherwise, compute for all fea-
tures.
nfeatures Number of features to mark as the top spatially variable.
assay Assay to pull the features (marks) from
image Name of image to pull the coordinates from

FindSubCluster Find subclusters under one cluster

Description
Find subclusters under one cluster

Usage
FindSubCluster(
object,
cluster,
graph.name,
subcluster.name = "sub.cluster",
resolution = 0.5,
algorithm = 1
)

Arguments
object An object
cluster the cluster to be sub-clustered
graph.name Name of graph to use for the clustering algorithm
subcluster.name
the name of sub cluster added in the meta.data
resolution Value of the resolution parameter, use a value above (below) 1.0 if you want to
obtain a larger (smaller) number of communities.
algorithm Algorithm for modularity optimization (1 = original Louvain algorithm; 2 =
Louvain algorithm with multilevel refinement; 3 = SLM algorithm; 4 = Leiden
algorithm). Leiden requires the leidenalg python.

Value
return a object with sub cluster labels in the sub-cluster.name variable
FindTransferAnchors 81

FindTransferAnchors Find transfer anchors

Description
Find a set of anchors between a reference and query object. These anchors can later be used to
transfer data from the reference to query object using the TransferData object.

Usage
FindTransferAnchors(
reference,
query,
normalization.method = "LogNormalize",
recompute.residuals = TRUE,
reference.assay = NULL,
reference.neighbors = NULL,
query.assay = NULL,
reduction = "pcaproject",
reference.reduction = NULL,
project.query = FALSE,
features = NULL,
scale = TRUE,
npcs = 30,
l2.norm = TRUE,
dims = 1:30,
k.anchor = 5,
k.filter = 200,
k.score = 30,
max.features = 200,
nn.method = "annoy",
n.trees = 50,
eps = 0,
approx.pca = TRUE,
mapping.score.k = NULL,
verbose = TRUE
)

Arguments
reference Seurat object to use as the reference
query Seurat object to use as the query
normalization.method
Name of normalization method used: LogNormalize or SCT.
recompute.residuals
If using SCT as a normalization method, compute query Pearson residuals using
the reference SCT model parameters.
82 FindTransferAnchors

reference.assay
Name of the Assay to use from reference
reference.neighbors
Name of the Neighbor to use from the reference. Optionally enables reuse of
precomputed neighbors.
query.assay Name of the Assay to use from query
reduction Dimensional reduction to perform when finding anchors. Options are:
• pcaproject: Project the PCA from the reference onto the query. We recom-
mend using PCA when reference and query datasets are from scRNA-seq
• lsiproject: Project the LSI from the reference onto the query. We recom-
mend using LSI when reference and query datasets are from scATAC-seq.
This requires that LSI has been computed for the reference dataset, and the
same features (eg, peaks or genome bins) are present in both the reference
and query. See RunTFIDF and RunSVD
• rpca: Project the PCA from the reference onto the query, and the PCA from
the query onto the reference (reciprocal PCA projection).
• cca: Run a CCA on the reference and query
reference.reduction
Name of dimensional reduction to use from the reference if running the pcapro-
ject workflow. Optionally enables reuse of precomputed reference dimensional
reduction. If NULL (default), use a PCA computed on the reference object.
project.query Project the PCA from the query dataset onto the reference. Use only in rare
cases where the query dataset has a much larger cell number, but the reference
dataset has a unique assay for transfer. In this case, the default features will
be set to the variable features of the query object that are alos present in the
reference.
features Features to use for dimensional reduction. If not specified, set as variable fea-
tures of the reference object which are also present in the query.
scale Scale query data.
npcs Number of PCs to compute on reference if reference.reduction is not provided.
l2.norm Perform L2 normalization on the cell embeddings after dimensional reduction
dims Which dimensions to use from the reduction to specify the neighbor search space
k.anchor How many neighbors (k) to use when finding anchors
k.filter How many neighbors (k) to use when filtering anchors. Set to NA to turn off
filtering.
k.score How many neighbors (k) to use when scoring anchors
max.features The maximum number of features to use when specifying the neighborhood
search space in the anchor filtering
nn.method Method for nearest neighbor finding. Options include: rann, annoy
n.trees More trees gives higher precision when using annoy approximate nearest neigh-
bor search
eps Error bound on the neighbor finding algorithm (from RANN or RcppAnnoy)
approx.pca Use truncated singular value decomposition to approximate PCA
FindTransferAnchors 83

mapping.score.k
Compute and store nearest k query neighbors in the AnchorSet object that is
returned. You can optionally set this if you plan on computing the mapping
score and want to enable reuse of some downstream neighbor calculations to
make the mapping score function more efficient.
verbose Print progress bars and output

Details

The main steps of this procedure are outlined below. For a more detailed description of the method-
ology, please see Stuart, Butler, et al Cell 2019. doi: 10.1016/j.cell.2019.05.031; doi: 10.1101/
460147

• Perform dimensional reduction. Exactly what is done here depends on the values set for the
reduction and project.query parameters. If reduction = "pcaproject", a PCA is per-
formed on either the reference (if project.query = FALSE) or the query (if project.query
= TRUE), using the features specified. The data from the other dataset is then projected onto
this learned PCA structure. If reduction = "cca", then CCA is performed on the reference
and query for this dimensional reduction step. If reduction = "lsiproject", the stored LSI
dimension reduction in the reference object is used to project the query dataset onto the refer-
ence. If l2.norm is set to TRUE, perform L2 normalization of the embedding vectors.
• Identify anchors between the reference and query - pairs of cells from each dataset that are
contained within each other’s neighborhoods (also known as mutual nearest neighbors).
• Filter low confidence anchors to ensure anchors in the low dimension space are in broad
agreement with the high dimensional measurements. This is done by looking at the neighbors
of each query cell in the reference dataset using max.features to define this space. If the
reference cell isn’t found within the first k.filter neighbors, remove the anchor.
• Assign each remaining anchor a score. For each anchor cell, determine the nearest k.score
anchors within its own dataset and within its pair’s dataset. Based on these neighborhoods,
construct an overall neighbor graph and then compute the shared neighbor overlap between
anchor and query cells (analogous to an SNN graph). We use the 0.01 and 0.90 quantiles on
these scores to dampen outlier effects and rescale to range between 0-1.

Value

Returns an AnchorSet object that can be used as input to TransferData, IntegrateEmbeddings

and MapQuery. The dimension reduction used for finding anchors is stored in the AnchorSet ob-
ject and can be used for computing anchor weights in downstream functions. Note that only the
requested dimensions are stored in the dimension reduction object in the AnchorSet. This means
that if dims=2:20 is used, for example, the dimension of the stored reduction is 1:19.

References

Stuart T, Butler A, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019;177:1888-1902

doi: 10.1016/j.cell.2019.05.031;
84 FindVariableFeatures

Examples
## Not run:
# to install the SeuratData package see https://github.com/satijalab/seurat-data
library(SeuratData)
data("pbmc3k")

# for demonstration, split the object into reference and query

pbmc.reference <- pbmc3k[, 1:1350]
pbmc.query <- pbmc3k[, 1351:2700]

# perform standard preprocessing on each object

pbmc.reference <- NormalizeData(pbmc.reference)
pbmc.reference <- FindVariableFeatures(pbmc.reference)
pbmc.reference <- ScaleData(pbmc.reference)

pbmc.query <- NormalizeData(pbmc.query)

pbmc.query <- FindVariableFeatures(pbmc.query)
pbmc.query <- ScaleData(pbmc.query)

# find anchors
anchors <- FindTransferAnchors(reference = pbmc.reference, query = pbmc.query)

# transfer labels
predictions <- TransferData(
anchorset = anchors,
refdata = pbmc.reference$seurat_annotations
)
pbmc.query <- AddMetaData(object = pbmc.query, metadata = predictions)

## End(Not run)

FindVariableFeatures Find variable features

Description
Identifies features that are outliers on a ’mean variability plot’.

Usage
FindVariableFeatures(object, ...)

## Default S3 method:
FindVariableFeatures(
object,
selection.method = "vst",
loess.span = 0.3,
clip.max = "auto",
FindVariableFeatures 85

mean.function = FastExpMean,
dispersion.function = FastLogVMR,
num.bin = 20,
binning.method = "equal_width",
verbose = TRUE,
...
)

## S3 method for class 'Assay'

FindVariableFeatures(
object,
selection.method = "vst",
loess.span = 0.3,
clip.max = "auto",
mean.function = FastExpMean,
dispersion.function = FastLogVMR,
num.bin = 20,
binning.method = "equal_width",
nfeatures = 2000,
mean.cutoff = c(0.1, 8),
dispersion.cutoff = c(1, Inf),
verbose = TRUE,
...
)

## S3 method for class 'SCTAssay'

FindVariableFeatures(object, nfeatures = 2000, ...)

## S3 method for class 'Seurat'

FindVariableFeatures(
object,
assay = NULL,
selection.method = "vst",
loess.span = 0.3,
clip.max = "auto",
mean.function = FastExpMean,
dispersion.function = FastLogVMR,
num.bin = 20,
binning.method = "equal_width",
nfeatures = 2000,
mean.cutoff = c(0.1, 8),
dispersion.cutoff = c(1, Inf),
verbose = TRUE,
...
)

Arguments
object An object
86 FindVariableFeatures

... Arguments passed to other methods

selection.method
How to choose top variable features. Choose one of :
• vst: First, fits a line to the relationship of log(variance) and log(mean) using
local polynomial regression (loess). Then standardizes the feature values
using the observed mean and expected variance (given by the fitted line).
Feature variance is then calculated on the standardized values after clipping
to a maximum (see clip.max parameter).
• mean.var.plot (mvp): First, uses a function to calculate average expres-
sion (mean.function) and dispersion (dispersion.function) for each feature.
Next, divides features into num.bin (deafult 20) bins based on their average
expression, and calculates z-scores for dispersion within each bin. The pur-
pose of this is to identify variable features while controlling for the strong
relationship between variability and average expression.
• dispersion (disp): selects the genes with the highest dispersion values
loess.span (vst method) Loess span parameter used when fitting the variance-mean rela-
tionship
clip.max (vst method) After standardization values larger than clip.max will be set to
clip.max; default is ’auto’ which sets this value to the square root of the number
of cells
mean.function Function to compute x-axis value (average expression). Default is to take the
mean of the detected (i.e. non-zero) values
dispersion.function
Function to compute y-axis value (dispersion). Default is to take the standard
deviation of all values
num.bin Total number of bins to use in the scaled analysis (default is 20)
binning.method Specifies how the bins should be computed. Available methods are:
• equal_width: each bin is of equal width along the x-axis [default]
• equal_frequency: each bin contains an equal number of features (can in-
crease statistical power to detect overdispersed features at high expression
values, at the cost of reduced resolution along the x-axis)
verbose show progress bar for calculations
nfeatures Number of features to select as top variable features; only used when selection.method
is set to 'dispersion' or 'vst'
mean.cutoff A two-length numeric vector with low- and high-cutoffs for feature means
dispersion.cutoff
A two-length numeric vector with low- and high-cutoffs for feature dispersions
assay Assay to use

Details
For the mean.var.plot method: Exact parameter settings may vary empirically from dataset to
dataset, and based on visual inspection of the plot. Setting the y.cutoff parameter to 2 identifies
features that are more than two standard deviations away from the average dispersion within a bin.
FoldChange 87

The default X-axis function is the mean expression level, and for Y-axis it is the log(Variance/mean).
All mean/variance calculations are not performed in log-space, but the results are reported in log-
space - see relevant functions for exact details.

FoldChange Fold Change

Description
Calculate log fold change and percentage of cells expressing each feature for different identity
classes.

Usage
FoldChange(object, ...)

## Default S3 method:
FoldChange(object, cells.1, cells.2, mean.fxn, fc.name, features = NULL, ...)

## S3 method for class 'Assay'

FoldChange(
object,
cells.1,
cells.2,
features = NULL,
slot = "data",
pseudocount.use = 1,
fc.name = NULL,
mean.fxn = NULL,
base = 2,
...
)

## S3 method for class 'DimReduc'

FoldChange(
object,
cells.1,
cells.2,
features = NULL,
slot = NULL,
pseudocount.use = NULL,
fc.name = NULL,
mean.fxn = NULL,
...
)

## S3 method for class 'Seurat'

88 FoldChange

FoldChange(
object,
ident.1 = NULL,
ident.2 = NULL,
group.by = NULL,
subset.ident = NULL,
assay = NULL,
slot = "data",
reduction = NULL,
features = NULL,
pseudocount.use = 1,
mean.fxn = NULL,
base = 2,
fc.name = NULL,
...
)

Arguments
object A Seurat object
... Arguments passed to other methods
cells.1 Vector of cell names belonging to group 1
cells.2 Vector of cell names belonging to group 2
mean.fxn Function to use for fold change or average difference calculation
fc.name Name of the fold change, average difference, or custom function column in the
output data.frame
features Features to calculate fold change for. If NULL, use all features
slot Slot to pull data from
pseudocount.use
Pseudocount to add to averaged expression values when calculating logFC. 1 by
default.
base The base with respect to which logarithms are computed.
ident.1 Identity class to calculate fold change for; pass an object of class phylo or ’clus-
tertree’ to calculate fold change for a node in a cluster tree; passing ’clustertree’
requires BuildClusterTree to have been run
ident.2 A second identity class for comparison; if NULL, use all other cells for compari-
son; if an object of class phylo or ’clustertree’ is passed to ident.1, must pass
a node to calculate fold change for
group.by Regroup cells into a different identity class prior to calculating fold change (see
example in FindMarkers)
subset.ident Subset a particular identity class prior to regrouping. Only relevant if group.by
is set (see example in FindMarkers)
assay Assay to use in fold change calculation
reduction Reduction to use - will calculate average difference on cell embeddings
GetAssay 89

Details
If the slot is scale.data or a reduction is specified, average difference is returned instead of log
fold change and the column is named "avg_diff". Otherwise, log2 fold change is returned with
column named "avg_log2_FC".

Value
Returns a data.frame

See Also
FindMarkers

Examples
data("pbmc_small")
FoldChange(pbmc_small, ident.1 = 1)

GetAssay Get an Assay object from a given Seurat object.

Description
Get an Assay object from a given Seurat object.

Usage
GetAssay(object, ...)

## S3 method for class 'Seurat'

GetAssay(object, assay = NULL, ...)

Arguments
object An object
... Arguments passed to other methods
assay Assay to get

Value
Returns an Assay object

Examples
data("pbmc_small")
GetAssay(object = pbmc_small, assay = "RNA")
90 GetIntegrationData

GetImage.SlideSeq Get Image Data

Description
Get Image Data

Usage
## S3 method for class 'SlideSeq'
GetImage(object, mode = c("grob", "raster", "plotly", "raw"), ...)

## S3 method for class 'STARmap'

GetImage(object, mode = c("grob", "raster", "plotly", "raw"), ...)

## S3 method for class 'VisiumV1'

GetImage(object, mode = c("grob", "raster", "plotly", "raw"), ...)

Arguments
object An object
mode How to return the image; should accept one of “grob”, “raster”, “plotly”, or
“raw”
... Arguments passed to other methods

See Also
SeuratObject::GetImage

GetIntegrationData Get integration data

Description
Get integration data

Usage
GetIntegrationData(object, integration.name, slot)

Arguments
object Seurat object
integration.name
Name of integration object
slot Which slot in integration object to get
GetResidual 91

Value
Returns data from the requested slot within the integrated object

GetResidual Calculate pearson residuals of features not in the scale.data

Description
This function calls sctransform::get_residuals.

Usage
GetResidual(
object,
features,
assay = NULL,
umi.assay = NULL,
clip.range = NULL,
replace.value = FALSE,
na.rm = TRUE,
verbose = TRUE
)

Arguments
object A seurat object
features Name of features to add into the scale.data
assay Name of the assay of the seurat object generated by SCTransform
umi.assay Name of the assay of the seurat object containing UMI matrix and the default is
RNA
clip.range Numeric of length two specifying the min and max values the Pearson residual
will be clipped to
replace.value Recalculate residuals for all features, even if they are already present. Useful if
you want to change the clip.range.
na.rm For features where there is no feature model stored, return NA for residual value
in scale.data when na.rm = FALSE. When na.rm is TRUE, only return residuals
for features with a model stored for all cells.
verbose Whether to print messages and progress bars

Value
Returns a Seurat object containing Pearson residuals of added features in its scale.data

See Also
get_residuals
92 GetTissueCoordinates.SlideSeq

Examples
data("pbmc_small")
pbmc_small <- SCTransform(object = pbmc_small, variable.features.n = 20)
pbmc_small <- GetResidual(object = pbmc_small, features = c('MS4A1', 'TCL1A'))

GetTissueCoordinates.SlideSeq
Get Tissue Coordinates

Description
Get Tissue Coordinates

Usage
## S3 method for class 'SlideSeq'
GetTissueCoordinates(object, ...)

## S3 method for class 'STARmap'

GetTissueCoordinates(object, qhulls = FALSE, ...)

## S3 method for class 'VisiumV1'

GetTissueCoordinates(
object,
scale = "lowres",
cols = c("imagerow", "imagecol"),
...
)

Arguments
object An object
... Arguments passed to other methods
qhulls return qhulls instead of centroids
scale A factor to scale the coordinates by; choose from: ’tissue’, ’fiducial’, ’hires’,
’lowres’, or NULL for no scaling
cols Columns of tissue coordinates data.frame to pull

See Also
SeuratObject::GetTissueCoordinates
GetTransferPredictions 93

GetTransferPredictions
Get the predicted identity

Description

Utility function to easily pull out the name of the class with the maximum prediction. This is
useful if you’ve set prediction.assay = TRUE in TransferData and want to have a vector with
the predicted class.

Usage

GetTransferPredictions(
object,
assay = "predictions",
slot = "data",
score.filter = 0.75
)

Arguments

object Seurat object

assay Name of the assay holding the predictions
slot Slot of the assay in which the prediction scores are stored
score.filter Return "Unassigned" for any cell with a score less than this value

Value

Returns a vector of predicted class names

Examples

## Not run:
prediction.assay <- TransferData(anchorset = anchors, refdata = reference$class)
query[["predictions"]] <- prediction.assay
query$predicted.id <- GetTransferPredictions(query)

## End(Not run)
94 GroupCorrelation

Graph-class The Graph Class

Description
For more details, please see the documentation in SeuratObject

See Also
SeuratObject::Graph-class

GroupCorrelation Compute the correlation of features broken down by groups with an-
other covariate

Description
Compute the correlation of features broken down by groups with another covariate

Usage
GroupCorrelation(
object,
assay = NULL,
slot = "scale.data",
var = NULL,
group.assay = NULL,
min.cells = 5,
ngroups = 6,
do.plot = TRUE
)

Arguments
object Seurat object
assay Assay to pull the data from
slot Slot in the assay to pull feature expression data from (counts, data, or scale.data)
var Variable with which to correlate the features
group.assay Compute the gene groups based off the data in this assay.
min.cells Only compute for genes in at least this many cells
ngroups Number of groups to split into
do.plot Display the group correlation boxplot (via GroupCorrelationPlot)

Value
A Seurat object with the correlation stored in metafeatures
GroupCorrelationPlot 95

GroupCorrelationPlot Boxplot of correlation of a variable (e.g. number of UMIs) with ex-

pression data

Description
Boxplot of correlation of a variable (e.g. number of UMIs) with expression data

Usage
GroupCorrelationPlot(
object,
assay = NULL,
feature.group = "feature.grp",
cor = "nCount_RNA_cor"
)

Arguments
object Seurat object
assay Assay where the feature grouping info and correlations are stored
feature.group Name of the column in meta.features where the feature grouping info is stored
cor Name of the column in meta.features where correlation info is stored

Value
Returns a ggplot boxplot of correlations split by group

HoverLocator Hover Locator

Description
Get quick information from a scatterplot by hovering over points

Usage
HoverLocator(plot, information = NULL, axes = TRUE, dark.theme = FALSE, ...)

Arguments
plot A ggplot2 plot
information An optional dataframe or matrix of extra information to be displayed on hover
axes Display or hide x- and y-axes
dark.theme Plot using a dark theme?
... Extra parameters to be passed to layout
96 HTODemux

See Also
layout ggplot_build DimPlot FeaturePlot

Examples
## Not run:
data("pbmc_small")
plot <- DimPlot(object = pbmc_small)
HoverLocator(plot = plot, information = FetchData(object = pbmc_small, vars = 'percent.mito'))

## End(Not run)

HTODemux Demultiplex samples based on data from cell ’hashing’

Description
Assign sample-of-origin for each cell, annotate doublets.

Usage
HTODemux(
object,
assay = "HTO",
positive.quantile = 0.99,
init = NULL,
nstarts = 100,
kfunc = "clara",
nsamples = 100,
seed = 42,
verbose = TRUE
)

Arguments
object Seurat object. Assumes that the hash tag oligo (HTO) data has been added and
normalized.
assay Name of the Hashtag assay (HTO by default)
positive.quantile
The quantile of inferred ’negative’ distribution for each hashtag - over which the
cell is considered ’positive’. Default is 0.99
init Initial number of clusters for hashtags. Default is the # of hashtag oligo names
+ 1 (to account for negatives)
nstarts nstarts value for k-means clustering (for kfunc = "kmeans"). 100 by default
HTOHeatmap 97

kfunc Clustering function for initial hashtag grouping. Default is "clara" for fast k-
medoids clustering on large applications, also support "kmeans" for kmeans
clustering
nsamples Number of samples to be drawn from the dataset used for clustering, for kfunc
= "clara"
seed Sets the random seed. If NULL, seed is not set
verbose Prints the output

Value

The Seurat object with the following demultiplexed information stored in the meta data:

hash.maxID Name of hashtag with the highest signal

hash.secondID Name of hashtag with the second highest signal
hash.margin The difference between signals for hash.maxID and hash.secondID
classification Classification result, with doublets/multiplets named by the top two highest hashtags
classification.global Global classification result (singlet, doublet or negative)
hash.ID Classification result where doublet IDs are collapsed

See Also

HTOHeatmap

Examples

## Not run:
object <- HTODemux(object)

## End(Not run)

HTOHeatmap Hashtag oligo heatmap

Description

Draws a heatmap of hashtag oligo signals across singlets/doublets/negative cells. Allows for the
visualization of HTO demultiplexing results.
98 HTOHeatmap

Usage

HTOHeatmap(
object,
assay = "HTO",
classification = paste0(assay, "_classification"),
global.classification = paste0(assay, "_classification.global"),
ncells = 5000,
singlet.names = NULL,
raster = TRUE
)

Arguments

object Seurat object. Assumes that the hash tag oligo (HTO) data has been added and
normalized, and demultiplexing has been run with HTODemux().
assay Hashtag assay name.
classification The naming for metadata column with classification result from HTODemux().
global.classification
The slot for metadata column specifying a cell as singlet/doublet/negative.
ncells Number of cells to plot. Default is to choose 5000 cells by random subsampling,
to avoid having to draw exceptionally large heatmaps.
singlet.names Namings for the singlets. Default is to use the same names as HTOs.
raster If true, plot with geom_raster, else use geom_tile. geom_raster may look blurry
on some viewing applications such as Preview due to how the raster is interpo-
lated. Set this to FALSE if you are encountering that issue (note that plots may
take longer to produce/render).

Value

Returns a ggplot2 plot object.

See Also

HTODemux

Examples
## Not run:
object <- HTODemux(object)
HTOHeatmap(object)

## End(Not run)
HVFInfo.SCTAssay 99

HVFInfo.SCTAssay Get Variable Feature Information

Description

Get variable feature information from SCTAssay objects

Usage

## S3 method for class 'SCTAssay'

HVFInfo(object, selection.method, status = FALSE, ...)

Arguments

object An object
selection.method
Which method to pull. For HVFInfo and VariableFeatures, choose one from
one of the following:
• “vst”
• “sctransform” or “sct”
• “mean.var.plot”, “dispersion”, “mvp”, or “disp”
For SVFInfo and SpatiallyVariableFeatures, choose from:
• “markvariogram”
• “moransi”
status Add variable status to the resulting data frame
... Arguments passed to other methods

See Also

HVFInfo

Examples

# Get the HVF info directly from an SCTAssay object

pbmc_small <- SCTransform(pbmc_small)
HVFInfo(pbmc_small[["SCT"]], selection.method = 'sct')[1:5, ]
100 IntegrateData

IFeaturePlot Visualize features in dimensional reduction space interactively

Description
Visualize features in dimensional reduction space interactively

Usage
IFeaturePlot(object, feature, dims = c(1, 2), reduction = NULL, slot = "data")

Arguments
object Seurat object
feature Feature to plot
dims Dimensions to plot, must be a two-length numeric vector specifying x- and y-
dimensions
reduction Which dimensionality reduction to use. If not specified, first searches for umap,
then tsne, then pca
slot Which slot to pull expression data from?

Value
Returns the final plot as a ggplot object

IntegrateData Integrate data

Description
Perform dataset integration using a pre-computed AnchorSet.

Usage
IntegrateData(
anchorset,
new.assay.name = "integrated",
normalization.method = c("LogNormalize", "SCT"),
features = NULL,
features.to.integrate = NULL,
dims = 1:30,
k.weight = 100,
weight.reduction = NULL,
sd.weight = 1,
IntegrateData 101

sample.tree = NULL,
preserve.order = FALSE,
eps = 0,
verbose = TRUE
)

Arguments
anchorset An AnchorSet object generated by FindIntegrationAnchors
new.assay.name Name for the new assay containing the integrated data
normalization.method
Name of normalization method used: LogNormalize or SCT
features Vector of features to use when computing the PCA to determine the weights.
Only set if you want a different set from those used in the anchor finding process
features.to.integrate
Vector of features to integrate. By default, will use the features used in anchor
finding.
dims Number of dimensions to use in the anchor weighting procedure
k.weight Number of neighbors to consider when weighting anchors
weight.reduction
Dimension reduction to use when calculating anchor weights. This can be one
of:
• A string, specifying the name of a dimension reduction present in all objects
to be integrated
• A vector of strings, specifying the name of a dimension reduction to use for
each object to be integrated
• A vector of DimReduc objects, specifying the object to use for each object
in the integration
• NULL, in which case a new PCA will be calculated and used to calculate
anchor weights
Note that, if specified, the requested dimension reduction will only be used for
calculating anchor weights in the first merge between reference and query, as
the merged object will subsequently contain more cells than was in query, and
weights will need to be calculated for all cells in the object.
sd.weight Controls the bandwidth of the Gaussian kernel for weighting
sample.tree Specify the order of integration. Order of integration should be encoded in a ma-
trix, where each row represents one of the pairwise integration steps. Negative
numbers specify a dataset, positive numbers specify the integration results from
a given row (the format of the merge matrix included in the hclust function
output). For example: matrix(c(-2,1,-3,-1),ncol = 2) gives:
[,1] [,2]
[1,] -2 -3
[2,] 1 -1
102 IntegrateData

Which would cause dataset 2 and 3 to be integrated first, then the resulting object
integrated with dataset 1.
If NULL, the sample tree will be computed automatically.
preserve.order Do not reorder objects based on size for each pairwise integration.
eps Error bound on the neighbor finding algorithm (from RANN)
verbose Print progress bars and output

Details
The main steps of this procedure are outlined below. For a more detailed description of the method-
ology, please see Stuart, Butler, et al Cell 2019. doi: 10.1016/j.cell.2019.05.031; doi: 10.1101/
460147
For pairwise integration:

• Construct a weights matrix that defines the association between each query cell and each
anchor. These weights are computed as 1 - the distance between the query cell and the anchor
divided by the distance of the query cell to the k.weightth anchor multiplied by the anchor
score computed in FindIntegrationAnchors. We then apply a Gaussian kernel width a
bandwidth defined by sd.weight and normalize across all k.weight anchors.
• Compute the anchor integration matrix as the difference between the two expression matrices
for every pair of anchor cells
• Compute the transformation matrix as the product of the integration matrix and the weights
matrix.
• Subtract the transformation matrix from the original expression matrix.

For multiple dataset integration, we perform iterative pairwise integration. To determine the order
of integration (if not specified via sample.tree), we

• Define a distance between datasets as the total number of cells in the smaller dataset divided
by the total number of anchors between the two datasets.
• Compute all pairwise distances between datasets
• Cluster this distance matrix to determine a guide tree

Value
Returns a Seurat object with a new integrated Assay. If normalization.method = "LogNormalize",
the integrated data is returned to the data slot and can be treated as log-normalized, corrected data.
If normalization.method = "SCT", the integrated data is returned to the scale.data slot and can
be treated as centered, corrected Pearson residuals.

References
Stuart T, Butler A, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019;177:1888-1902
doi: 10.1016/j.cell.2019.05.031
IntegrateEmbeddings 103

Examples
## Not run:
# to install the SeuratData package see https://github.com/satijalab/seurat-data
library(SeuratData)
data("panc8")

# panc8 is a merged Seurat object containing 8 separate pancreas datasets

# split the object by dataset
pancreas.list <- SplitObject(panc8, split.by = "tech")

# perform standard preprocessing on each object

for (i in 1:length(pancreas.list)) {
pancreas.list[[i]] <- NormalizeData(pancreas.list[[i]], verbose = FALSE)
pancreas.list[[i]] <- FindVariableFeatures(
pancreas.list[[i]], selection.method = "vst",
nfeatures = 2000, verbose = FALSE
)
}

# find anchors
anchors <- FindIntegrationAnchors(object.list = pancreas.list)

# integrate data
integrated <- IntegrateData(anchorset = anchors)

## End(Not run)

IntegrateEmbeddings Integrate low dimensional embeddings

Description
Perform dataset integration using a pre-computed Anchorset of specified low dimensional represen-
tations.

Usage
IntegrateEmbeddings(anchorset, ...)

## S3 method for class 'IntegrationAnchorSet'

IntegrateEmbeddings(
anchorset,
new.reduction.name = "integrated_dr",
reductions = NULL,
dims.to.integrate = NULL,
k.weight = 100,
weight.reduction = NULL,
sd.weight = 1,
104 IntegrateEmbeddings

sample.tree = NULL,
preserve.order = FALSE,
verbose = TRUE,
...
)

## S3 method for class 'TransferAnchorSet'

IntegrateEmbeddings(
anchorset,
reference,
query,
new.reduction.name = "integrated_dr",
reductions = "pcaproject",
dims.to.integrate = NULL,
k.weight = 100,
weight.reduction = NULL,
reuse.weights.matrix = TRUE,
sd.weight = 1,
preserve.order = FALSE,
verbose = TRUE,
...
)

Arguments
anchorset An AnchorSet object
... Reserved for internal use
new.reduction.name
Name for new integrated dimensional reduction.
reductions Name of reductions to be integrated. For a TransferAnchorSet, this should be the
name of a reduction present in the anchorset object (for example, "pcaproject").
For an IntegrationAnchorSet, this should be a DimReduc object containing all
cells present in the anchorset object.
dims.to.integrate
Number of dimensions to return integrated values for
k.weight Number of neighbors to consider when weighting anchors
weight.reduction
Dimension reduction to use when calculating anchor weights. This can be one
of:
• A string, specifying the name of a dimension reduction present in all objects
to be integrated
• A vector of strings, specifying the name of a dimension reduction to use for
each object to be integrated
• A vector of DimReduc objects, specifying the object to use for each object
in the integration
• NULL, in which case the full corrected space is used for computing anchor
weights.
IntegrationAnchorSet-class 105

sd.weight Controls the bandwidth of the Gaussian kernel for weighting

sample.tree Specify the order of integration. Order of integration should be encoded in a ma-
trix, where each row represents one of the pairwise integration steps. Negative
numbers specify a dataset, positive numbers specify the integration results from
a given row (the format of the merge matrix included in the hclust function
output). For example: matrix(c(-2,1,-3,-1),ncol = 2) gives:
[,1] [,2]
[1,] -2 -3
[2,] 1 -1
Which would cause dataset 2 and 3 to be integrated first, then the resulting object
integrated with dataset 1.
If NULL, the sample tree will be computed automatically.
preserve.order Do not reorder objects based on size for each pairwise integration.
verbose Print progress bars and output
reference Reference object used in anchorset construction
query Query object used in anchorset construction
reuse.weights.matrix
Can be used in conjunction with the store.weights parameter in TransferData to
reuse a precomputed weights matrix.

Details
The main steps of this procedure are identical to IntegrateData with one key distinction. When
computing the weights matrix, the distance calculations are performed in the full space of integrated
embeddings when integrating more than two datasets, as opposed to a reduced PCA space which is
the default behavior in IntegrateData.

Value
When called on a TransferAnchorSet (from FindTransferAnchors), this will return the query object
with the integrated embeddings stored in a new reduction. When called on an IntegrationAnchorSet
(from IntegrateData), this will return a merged object with the integrated reduction stored.

IntegrationAnchorSet-class
The IntegrationAnchorSet Class

Description
Inherits from the Anchorset class. Implemented mainly for method dispatch purposes. See AnchorSet
for slot details.
106 ISpatialDimPlot

IntegrationData-class The IntegrationData Class

Description
The IntegrationData object is an intermediate storage container used internally throughout the inte-
gration procedure to hold bits of data that are useful downstream.

Slots
neighbors List of neighborhood information for cells (outputs of RANN::nn2)
weights Anchor weight matrix
integration.matrix Integration matrix
anchors Anchor matrix
offsets The offsets used to enable cell look up in downstream functions
objects.ncell Number of cells in each object in the object.list
sample.tree Sample tree used for ordering multi-dataset integration

ISpatialDimPlot Visualize clusters spatially and interactively

Description
Visualize clusters spatially and interactively

Usage
ISpatialDimPlot(object, image = NULL, group.by = NULL, alpha = c(0.3, 1))

Arguments
object Seurat object
image Name of the image to use in the plot
group.by Name of one or more metadata columns to group (color) cells by (for example,
orig.ident); pass ’ident’ to group by identity class
alpha Controls opacity of spots. Provide as a vector specifying the min and max for
SpatialFeaturePlot. For SpatialDimPlot, provide a single alpha value for each
plot.

Value
Returns final plot as a ggplot object
ISpatialFeaturePlot 107

ISpatialFeaturePlot Visualize features spatially and interactively

Description

Visualize features spatially and interactively

Usage

ISpatialFeaturePlot(
object,
feature,
image = NULL,
slot = "data",
alpha = c(0.1, 1)
)

Arguments

object Seurat object

feature Feature to visualize
image Name of the image to use in the plot
slot Which slot to pull expression data from?
alpha Controls opacity of spots. Provide as a vector specifying the min and max for
SpatialFeaturePlot. For SpatialDimPlot, provide a single alpha value for each
plot.

Value

Returns final plot as a ggplot object

JackStraw Determine statistical significance of PCA scores.

Description

Randomly permutes a subset of data, and calculates projected PCA scores for these ’random’ genes.
Then compares the PCA scores for the ’random’ genes with the observed PCA scores to determine
statistical signifance. End result is a p-value for each gene’s association with each principal com-
ponent.
108 JackStraw

Usage

JackStraw(
object,
reduction = "pca",
assay = NULL,
dims = 20,
num.replicate = 100,
prop.freq = 0.01,
verbose = TRUE,
maxit = 1000
)

Arguments

object Seurat object

reduction DimReduc to use. ONLY PCA CURRENTLY SUPPORTED.
assay Assay used to calculate reduction.
dims Number of PCs to compute significance for
num.replicate Number of replicate samplings to perform
prop.freq Proportion of the data to randomly permute for each replicate
verbose Print progress bar showing the number of replicates that have been processed.
maxit maximum number of iterations to be performed by the irlba function of RunPCA

Value

Returns a Seurat object where JS(object = object[[’pca’]], slot = ’empirical’) represents p-values
for each gene in the PCA analysis. If ProjectPCA is subsequently run, JS(object = object[[’pca’]],
slot = ’full’) then represents p-values for all genes.

References

Inspired by Chung et al, Bioinformatics (2014)

Examples
## Not run:
data("pbmc_small")
pbmc_small = suppressWarnings(JackStraw(pbmc_small))
head(JS(object = pbmc_small[['pca']], slot = 'empirical'))

## End(Not run)
JackStrawData-class 109

JackStrawData-class The JackStrawData Class

Description
For more details, please see the documentation in SeuratObject

See Also
SeuratObject::JackStrawData-class

JackStrawPlot JackStraw Plot

Description
Plots the results of the JackStraw analysis for PCA significance. For each PC, plots a QQ-plot
comparing the distribution of p-values for all genes across each PC, compared with a uniform
distribution. Also determines a p-value for the overall significance of each PC (see Details).

Usage
JackStrawPlot(
object,
dims = 1:5,
cols = NULL,
reduction = "pca",
xmax = 0.1,
ymax = 0.3
)

Arguments
object Seurat object
dims Dims to plot
cols Vector of colors, each color corresponds to an individual PC. This may also
be a single character or numeric value corresponding to a palette as specified
by brewer.pal.info. By default, ggplot2 assigns colors. We also include a
number of palettes from the pals package. See DiscretePalette for details.
reduction reduction to pull jackstraw info from
xmax X-axis maximum on each QQ plot.
ymax Y-axis maximum on each QQ plot.
110 L2CCA

Details

Significant PCs should show a p-value distribution (black curve) that is strongly skewed to the left
compared to the null distribution (dashed line) The p-value for each PC is based on a proportion test
comparing the number of genes with a p-value below a particular threshold (score.thresh), compared
with the proportion of genes expected under a uniform distribution of p-values.

Value

A ggplot object

Author(s)

Omri Wurtzel

See Also

ScoreJackStraw

Examples
data("pbmc_small")
JackStrawPlot(object = pbmc_small)

L2CCA L2-Normalize CCA

Description

Perform l2 normalization on CCs

Usage

L2CCA(object, ...)

Arguments

object Seurat object

... Additional parameters to L2Dim.
L2Dim 111

L2Dim L2-normalization

Description
Perform l2 normalization on given dimensional reduction

Usage
L2Dim(object, reduction, new.dr = NULL, new.key = NULL)

Arguments
object Seurat object
reduction Dimensional reduction to normalize
new.dr name of new dimensional reduction to store (default is olddr.l2)
new.key name of key for new dimensional reduction

Value
Returns a Seurat object

LabelClusters Label clusters on a ggplot2-based scatter plot

Description
Label clusters on a ggplot2-based scatter plot

Usage
LabelClusters(
plot,
id,
clusters = NULL,
labels = NULL,
split.by = NULL,
repel = TRUE,
box = FALSE,
geom = "GeomPoint",
position = "median",
...
)
112 LabelPoints

Arguments
plot A ggplot2-based scatter plot
id Name of variable used for coloring scatter plot
clusters Vector of cluster ids to label
labels Custom labels for the clusters
split.by Split labels by some grouping label, useful when using facet_wrap or facet_grid
repel Use geom_text_repel to create nicely-repelled labels
box Use geom_label/geom_label_repel (includes a box around the text labels)
geom Name of geom to get X/Y aesthetic names for
position How to place the label if repel = FALSE. If "median", place the label at the
median position. If "nearest" place the label at the position of the nearest data
point to the median.
... Extra parameters to geom_text_repel, such as size

Value
A ggplot2-based scatter plot with cluster labels

See Also
geom_text_repel geom_text

Examples
data("pbmc_small")
plot <- DimPlot(object = pbmc_small)
LabelClusters(plot = plot, id = 'ident')

LabelPoints Add text labels to a ggplot2 plot

Description
Add text labels to a ggplot2 plot

Usage
LabelPoints(
plot,
points,
labels = NULL,
repel = FALSE,
xnudge = 0.3,
ynudge = 0.05,
...
)
LinkedPlots 113

Arguments
plot A ggplot2 plot with a GeomPoint layer
points A vector of points to label; if NULL, will use all points in the plot
labels A vector of labels for the points; if NULL, will use rownames of the data provided
to the plot at the points selected
repel Use geom_text_repel to create a nicely-repelled labels; this is slow when a lot
of points are being plotted. If using repel, set xnudge and ynudge to 0
xnudge, ynudge Amount to nudge X and Y coordinates of labels by
... Extra parameters passed to geom_text

Value
A ggplot object

See Also
geom_text

Examples
data("pbmc_small")
ff <- TopFeatures(object = pbmc_small[['pca']])
cc <- TopCells(object = pbmc_small[['pca']])
plot <- FeatureScatter(object = pbmc_small, feature1 = ff[1], feature2 = ff[2])
LabelPoints(plot = plot, points = cc)

LinkedPlots Visualize spatial and clustering (dimensional reduction) data in a

linked, interactive framework

Description
Visualize spatial and clustering (dimensional reduction) data in a linked, interactive framework

Usage
LinkedDimPlot(
object,
dims = 1:2,
reduction = NULL,
image = NULL,
group.by = NULL,
alpha = c(0.1, 1),
combine = TRUE
)
114 LinkedPlots

LinkedFeaturePlot(
object,
feature,
dims = 1:2,
reduction = NULL,
image = NULL,
slot = "data",
alpha = c(0.1, 1),
combine = TRUE
)

Arguments

object Seurat object

dims Dimensions to plot, must be a two-length numeric vector specifying x- and y-
dimensions
reduction Which dimensionality reduction to use. If not specified, first searches for umap,
then tsne, then pca
image Name of the image to use in the plot
group.by Name of one or more metadata columns to group (color) cells by (for example,
orig.ident); pass ’ident’ to group by identity class
alpha Controls opacity of spots. Provide as a vector specifying the min and max for
SpatialFeaturePlot. For SpatialDimPlot, provide a single alpha value for each
plot.
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot objects
feature Feature to visualize
slot Which slot to pull expression data from?

Value

Returns final plots. If combine, plots are stiched together using CombinePlots; otherwise, returns
a list of ggplot objects

Examples
## Not run:
LinkedDimPlot(seurat.object)
LinkedFeaturePlot(seurat.object, feature = 'Hpca')

## End(Not run)
Load10X_Spatial 115

Load10X_Spatial Load a 10x Genomics Visium Spatial Experiment into a Seurat object

Description
Load a 10x Genomics Visium Spatial Experiment into a Seurat object

Usage
Load10X_Spatial(
data.dir,
filename = "filtered_feature_bc_matrix.h5",
assay = "Spatial",
slice = "slice1",
filter.matrix = TRUE,
to.upper = FALSE,
image = NULL,
...
)

Arguments
data.dir Directory containing the H5 file specified by filename and the image data in a
subdirectory called spatial
filename Name of H5 file containing the feature barcode matrix
assay Name of the initial assay
slice Name for the stored image of the tissue slice
filter.matrix Only keep spots that have been determined to be over tissue
to.upper Converts all feature names to upper case. This can provide an approximate
conversion of mouse to human gene names which can be useful in an explorative
analysis. For cross-species comparisons, orthologous genes should be identified
across species and used instead.
image An object of class VisiumV1. Typically, an output from Read10X_Image
... Arguments passed to Read10X_h5

Value
A Seurat object

Examples
## Not run:
data_dir <- 'path/to/data/directory'
list.files(data_dir) # Should show filtered_feature_bc_matrix.h5
Load10X_Spatial(data.dir = data_dir)
116 LoadSTARmap

## End(Not run)

LoadAnnoyIndex Load the Annoy index file

Description
Load the Annoy index file

Usage
LoadAnnoyIndex(object, file)

Arguments
object Neighbor object
file Path to file with annoy index

Value
Returns the Neighbor object with the index stored

LoadSTARmap Load STARmap data

Description
Load STARmap data

Usage
LoadSTARmap(
data.dir,
counts.file = "cell_barcode_count.csv",
gene.file = "genes.csv",
qhull.file = "qhulls.tsv",
centroid.file = "centroids.tsv",
assay = "Spatial",
image = "image"
)
LocalStruct 117

Arguments

data.dir location of data directory that contains the counts matrix, gene name, qhull, and
centroid files.
counts.file name of file containing the counts matrix (csv)
gene.file name of file containing the gene names (csv)
qhull.file name of file containing the hull coordinates (tsv)
centroid.file name of file containing the centroid positions (tsv)
assay Name of assay to associate spatial data to
image Name of "image" object storing spatial coordinates

Value

A Seurat object

See Also

STARmap

LocalStruct Calculate the local structure preservation metric

Description

Calculates a metric that describes how well the local structure of each group prior to integration
is preserved after integration. This procedure works as follows: For each group, compute a PCA,
compute the top num.neighbors in pca space, compute the top num.neighbors in corrected pca
space, compute the size of the intersection of those two sets of neighbors. Return the average over
all groups.

Usage

LocalStruct(
object,
grouping.var,
idents = NULL,
neighbors = 100,
reduction = "pca",
reduced.dims = 1:10,
orig.dims = 1:10,
verbose = TRUE
)
118 LogNormalize

Arguments
object Seurat object
grouping.var Grouping variable
idents Optionally specify a set of idents to compute metric for
neighbors Number of neighbors to compute in pca/corrected pca space
reduction Dimensional reduction to use for corrected space
reduced.dims Number of reduced dimensions to use
orig.dims Number of PCs to use in original space
verbose Display progress bar

Value
Returns the average preservation metric

LogNormalize Normalize raw data

Description
Normalize count data per cell and transform to log scale

Usage
LogNormalize(data, scale.factor = 10000, verbose = TRUE)

Arguments
data Matrix with the raw count data
scale.factor Scale the data. Default is 1e4
verbose Print progress

Value
Returns a matrix with the normalize and log transformed data

Examples
mat <- matrix(data = rbinom(n = 25, size = 5, prob = 0.2), nrow = 5)
mat
mat_norm <- LogNormalize(data = mat)
mat_norm
LogVMR 119

LogVMR Calculate the variance to mean ratio of logged values

Description

Calculate the variance to mean ratio (VMR) in non-logspace (return answer in log-space)

Usage

LogVMR(x, ...)

Arguments

x A vector of values
... Other arguments (not used)

Value

Returns the VMR in log-space

Examples

LogVMR(x = c(1, 2, 3))

MappingScore Metric for evaluating mapping success

Description

This metric was designed to help identify query cells that aren’t well represented in the reference
dataset. The intuition for the score is that we are going to project the query cells into a reference-
defined space and then project them back onto the query. By comparing the neighborhoods before
and after projection, we identify cells who’s local neighborhoods are the most affected by this
transformation. This could be because there is a population of query cells that aren’t present in the
reference or the state of the cells in the query is significantly different from the equivalent cell type
in the reference.
120 MappingScore

Usage
MappingScore(anchors, ...)

## Default S3 method:
MappingScore(
anchors,
combined.object,
query.neighbors,
ref.embeddings,
query.embeddings,
kanchors = 50,
ndim = 50,
ksmooth = 100,
ksnn = 20,
snn.prune = 0,
subtract.first.nn = TRUE,
nn.method = "annoy",
n.trees = 50,
query.weights = NULL,
verbose = TRUE,
...
)

## S3 method for class 'AnchorSet'

MappingScore(
anchors,
kanchors = 50,
ndim = 50,
ksmooth = 100,
ksnn = 20,
snn.prune = 0,
subtract.first.nn = TRUE,
nn.method = "annoy",
n.trees = 50,
query.weights = NULL,
verbose = TRUE,
...
)

Arguments
anchors AnchorSet object or just anchor matrix from the Anchorset object returned from
FindTransferAnchors
... Reserved for internal use
combined.object
Combined object (ref + query) from the Anchorset object returned
query.neighbors
Neighbors object computed on query cells
MapQuery 121

ref.embeddings Reference embeddings matrix

query.embeddings
Query embeddings matrix
kanchors Number of anchors to use in projection steps when computing weights
ndim Number of dimensions to use when working with low dimensional projections
of the data
ksmooth Number of cells to average over when computing transition probabilities
ksnn Number of cells to average over when determining the kernel bandwidth from
the SNN graph
snn.prune Amount of pruning to apply to edges in SNN graph
subtract.first.nn
Option to the scoring function when computing distances to subtract the distance
to the first nearest neighbor
nn.method Nearest neighbor method to use (annoy or RANN)
n.trees More trees gives higher precision when using annoy approximate nearest neigh-
bor search
query.weights Query weights matrix for reuse
verbose Display messages/progress

Value
Returns a vector of cell scores

MapQuery Map query cells to a reference

Description
This is a convenience wrapper function around the following three functions that are often run to-
gether when mapping query data to a reference: TransferData, IntegrateEmbeddings, ProjectUMAP.
Note that by default, the weight.reduction parameter for all functions will be set to the dimen-
sion reduction method used in the FindTransferAnchors function call used to construct the anchor
object, and the dims parameter will be the same dimensions used to find anchors.

Usage
MapQuery(
anchorset,
query,
reference,
refdata = NULL,
new.reduction.name = NULL,
reference.reduction = NULL,
reference.dims = NULL,
122 MapQuery

query.dims = NULL,
reduction.model = NULL,
transferdata.args = list(),
integrateembeddings.args = list(),
projectumap.args = list(),
verbose = TRUE
)

Arguments
anchorset An AnchorSet object
query Query object used in anchorset construction
reference Reference object used in anchorset construction
refdata Data to transfer. This can be specified in one of two ways:
• The reference data itself as either a vector where the names correspond to
the reference cells, or a matrix, where the column names correspond to the
reference cells.
• The name of the metadata field or assay from the reference object provided.
This requires the reference parameter to be specified. If pulling assay data
in this manner, it will pull the data from the data slot. To transfer data from
other slots, please pull the data explicitly with GetAssayData and provide
that matrix here.
new.reduction.name
Name for new integrated dimensional reduction.
reference.reduction
Name of reduction to use from the reference for neighbor finding
reference.dims Dimensions (columns) to use from reference
query.dims Dimensions (columns) to use from query
reduction.model
DimReduc object that contains the umap model
transferdata.args
A named list of additional arguments to TransferData
integrateembeddings.args
A named list of additional arguments to IntegrateEmbeddings
projectumap.args
A named list of additional arguments to ProjectUMAP
verbose Print progress bars and output

Value
Returns a modified query Seurat object containing:
• New Assays corresponding to the features transferred and/or their corresponding prediction
scores from TransferData
• An integrated reduction from IntegrateEmbeddings
• A projected UMAP reduction of the query cells projected into the reference UMAP using
ProjectUMAP
merge.SCTAssay 123

merge.SCTAssay Merge SCTAssay objects

Description

Merge SCTAssay objects

Usage

## S3 method for class 'SCTAssay'

merge(
x = NULL,
y = NULL,
add.cell.ids = NULL,
merge.data = TRUE,
na.rm = TRUE,
...
)

Arguments

x A Seurat object
y A single Seurat object or a list of Seurat objects
add.cell.ids A character vector of length(x = c(x,y)); appends the corresponding values
to the start of each objects’ cell names
merge.data Merge the data slots instead of just merging the counts (which requires renormal-
ization); this is recommended if the same normalization approach was applied
to all objects
na.rm If na.rm = TRUE, this will only preserve residuals that are present in all SCTAs-
says being merged. Otherwise, missing residuals will be populated with NAs.
... Arguments passed to other methods

MetaFeature Aggregate expression of multiple features into a single feature

Description

Calculates relative contribution of each feature to each cell for given set of features.
124 MinMax

Usage
MetaFeature(
object,
features,
meta.name = "metafeature",
cells = NULL,
assay = NULL,
slot = "data"
)

Arguments
object A Seurat object
features List of features to aggregate
meta.name Name of column in metadata to store metafeature
cells List of cells to use (default all cells)
assay Which assay to use
slot Which slot to take data from (default data)

Value
Returns a Seurat object with metafeature stored in objct metadata

Examples
data("pbmc_small")
pbmc_small <- MetaFeature(
object = pbmc_small,
features = c("LTB", "EAF2"),
meta.name = 'var.aggregate'
)
head(pbmc_small[[]])

MinMax Apply a ceiling and floor to all values in a matrix

Description
Apply a ceiling and floor to all values in a matrix

Usage
MinMax(data, min, max)
MixingMetric 125

Arguments
data Matrix or data frame
min all values below this min value will be replaced with min
max all values above this max value will be replaced with max

Value
Returns matrix after performing these floor and ceil operations

Examples
mat <- matrix(data = rbinom(n = 25, size = 20, prob = 0.2 ), nrow = 5)
mat
MinMax(data = mat, min = 4, max = 5)

MixingMetric Calculates a mixing metric

Description
Here we compute a measure of how well mixed a composite dataset is. To compute, we first
examine the local neighborhood for each cell (looking at max.k neighbors) and determine for each
group (could be the dataset after integration) the k nearest neighbor and what rank that neighbor
was in the overall neighborhood. We then take the median across all groups as the mixing metric
per cell.

Usage
MixingMetric(
object,
grouping.var,
reduction = "pca",
dims = 1:2,
k = 5,
max.k = 300,
eps = 0,
verbose = TRUE
)

Arguments
object Seurat object
grouping.var Grouping variable for dataset
reduction Which dimensionally reduced space to use
dims Dimensions to use
126 MixscapeHeatmap

k Neighbor number to examine per group

max.k Maximum size of local neighborhood to compute
eps Error bound on the neighbor finding algorithm (from RANN)
verbose Displays progress bar

Value
Returns a vector of values of the mixing metric for each cell

MixscapeHeatmap Differential expression heatmap for mixscape

Description
Draws a heatmap of single cell feature expression with cells ordered by their mixscape ko probabil-
ities.

Usage
MixscapeHeatmap(
object,
ident.1 = NULL,
ident.2 = NULL,
balanced = TRUE,
logfc.threshold = 0.25,
assay = "RNA",
max.genes = 100,
test.use = "wilcox",
max.cells.group = NULL,
order.by.prob = TRUE,
group.by = NULL,
mixscape.class = "mixscape_class",
prtb.type = "KO",
fc.name = "avg_log2FC",
pval.cutoff = 0.05,
...
)

Arguments
object An object
ident.1 Identity class to define markers for; pass an object of class phylo or ’clus-
tertree’ to find markers for a node in a cluster tree; passing ’clustertree’ requires
BuildClusterTree to have been run
ident.2 A second identity class for comparison; if NULL, use all other cells for compari-
son; if an object of class phylo or ’clustertree’ is passed to ident.1, must pass
a node to find markers for
MixscapeHeatmap 127

balanced Plot an equal number of genes with both groups of cells.

logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.
assay Assay to use in differential expression testing
max.genes Total number of DE genes to plot.
test.use Denotes which test to use. Available options are:
• "wilcox" : Identifies differentially expressed genes between two groups of
cells using a Wilcoxon Rank Sum test (default)
• "bimod" : Likelihood-ratio test for single cell gene expression, (McDavid
et al., Bioinformatics, 2013)
• "roc" : Identifies ’markers’ of gene expression using ROC analysis. For
each gene, evaluates (using AUC) a classifier built on that gene alone, to
classify between two groups of cells. An AUC value of 1 means that ex-
pression values for this gene alone can perfectly classify the two groupings
(i.e. Each of the cells in cells.1 exhibit a higher level than each of the cells
in cells.2). An AUC value of 0 also means there is perfect classification, but
in the other direction. A value of 0.5 implies that the gene has no predictive
power to classify the two groups. Returns a ’predictive power’ (abs(AUC-
0.5) * 2) ranked matrix of putative differentially expressed genes.
• "t" : Identify differentially expressed genes between two groups of cells
using the Student’s t-test.
• "negbinom" : Identifies differentially expressed genes between two groups
of cells using a negative binomial generalized linear model. Use only for
UMI-based datasets
• "poisson" : Identifies differentially expressed genes between two groups of
cells using a poisson generalized linear model. Use only for UMI-based
datasets
• "LR" : Uses a logistic regression framework to determine differentially
expressed genes. Constructs a logistic regression model predicting group
membership based on each feature individually and compares this to a null
model with a likelihood ratio test.
• "MAST" : Identifies differentially expressed genes between two groups of
cells using a hurdle model tailored to scRNA-seq data. Utilizes the MAST
package to run the DE testing.
• "DESeq2" : Identifies differentially expressed genes between two groups of
cells based on a model using DESeq2 which uses a negative binomial distri-
bution (Love et al, Genome Biology, 2014).This test does not support pre-
filtering of genes based on average difference (or percent detection rate) be-
tween cell groups. However, genes may be pre-filtered based on their min-
imum detection rate (min.pct) across both cell groups. To use this method,
please install DESeq2, using the instructions at https://bioconductor.org/packages/release/bioc/html/D
max.cells.group
Number of cells per identity to plot.
128 MixscapeLDA

order.by.prob Order cells on heatmap based on their mixscape knockout probability from high-
est to lowest score.
group.by (Deprecated) Option to split densities based on mixscape classification. Please
use mixscape.class instead
mixscape.class metadata column with mixscape classifications.
prtb.type specify type of CRISPR perturbation expected for labeling mixscape classifica-
tions. Default is KO.
fc.name Name of the fold change, average difference, or custom function column in the
output data.frame. Default is avg_log2FC
pval.cutoff P-value cut-off for selection of significantly DE genes.
... Arguments passed to other methods and to specific DE methods

Value

A ggplot object.

MixscapeLDA Linear discriminant analysis on pooled CRISPR screen data.

Description

This function performs unsupervised PCA on each mixscape class separately and projects each
subspace onto all cells in the data. Finally, it uses the first 10 principle components from each
projection as input to lda in MASS package together with mixscape class labels.

Usage

MixscapeLDA(
object,
assay = NULL,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.key = "LDA_",
seed = 42,
pc.assay = "PRTB",
labels = "gene",
nt.label = "NT",
npcs = 10,
verbose = TRUE,
logfc.threshold = 0.25
)
ModalityWeights-class 129

Arguments
object An object of class Seurat.
assay Assay to use for performing Linear Discriminant Analysis (LDA).
ndims.print Number of LDA dimensions to print.
nfeatures.print
Number of features to print for each LDA component.
reduction.key Reduction key name.
seed Value for random seed
pc.assay Assay to use for running Principle components analysis.
labels Meta data column with target gene class labels.
nt.label Name of non-targeting cell class.
npcs Number of principle components to use.
verbose Print progress bar.
logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.

Value
Returns a Seurat object with LDA added in the reduction slot.

ModalityWeights-class The ModalityWeights Class

Description
The ModalityWeights class is an intermediate data storage class that stores the modality weight and
other related information needed for performing downstream analyses - namely data integration
(FindModalityWeights) and data transfer (FindMultiModalNeighbors).

Slots
modality.weight.list A list of modality weights value from all modalities
modality.assay Names of assays for the list of dimensional reductions
params A list of parameters used in the FindModalityWeights
score.matrix a list of score matrices representing cross and within-modality prediction score, and
kernel value
command Store log of parameters that were used
130 MULTIseqDemux

MULTIseqDemux Demultiplex samples based on classification method from MULTI-seq

(McGinnis et al., bioRxiv 2018)

Description
Identify singlets, doublets and negative cells from multiplexing experiments. Annotate singlets by
tags.

Usage
MULTIseqDemux(
object,
assay = "HTO",
quantile = 0.7,
autoThresh = FALSE,
maxiter = 5,
qrange = seq(from = 0.1, to = 0.9, by = 0.05),
verbose = TRUE
)

Arguments
object Seurat object. Assumes that the specified assay data has been added
assay Name of the multiplexing assay (HTO by default)
quantile The quantile to use for classification
autoThresh Whether to perform automated threshold finding to define the best quantile. De-
fault is FALSE
maxiter Maximum number of iterations if autoThresh = TRUE. Default is 5
qrange A range of possible quantile values to try if autoThresh = TRUE
verbose Prints the output

Value
A Seurat object with demultiplexing results stored at object$MULTI_ID

References
https://www.biorxiv.org/content/10.1101/387241v1

Examples
## Not run:
object <- MULTIseqDemux(object)

## End(Not run)
Neighbor-class 131

Neighbor-class The Neighbor Class

Description
For more details, please see the documentation in SeuratObject

See Also
SeuratObject::Neighbor-class

NNPlot Highlight Neighbors in DimPlot

Description
It will color the query cells and the neighbors of the query cells in the DimPlot

Usage
NNPlot(
object,
reduction,
nn.idx,
query.cells,
dims = 1:2,
label = FALSE,
label.size = 4,
repel = FALSE,
sizes.highlight = 2,
pt.size = 1,
cols.highlight = c("#377eb8", "#e41a1c"),
na.value = "#bdbdbd",
order = c("self", "neighbors", "other"),
show.all.cells = TRUE,
...
)

Arguments
object Seurat object
reduction Which dimensionality reduction to use. If not specified, first searches for umap,
then tsne, then pca
nn.idx the neighbor index of all cells
132 NormalizeData

query.cells cells used to find their neighbors

dims Dimensions to plot, must be a two-length numeric vector specifying x- and y-
dimensions
label Whether to label the clusters
label.size Sets size of labels
repel Repel labels
sizes.highlight
Size of highlighted cells; will repeat to the length groups in cells.highlight
pt.size Adjust point size for plotting
cols.highlight A vector of colors to highlight the cells as; will repeat to the length groups in
cells.highlight
na.value Color value for NA points when using custom scale
order Specify the order of plotting for the idents. This can be useful for crowded plots
if points of interest are being buried. Provide either a full list of valid idents or a
subset to be plotted last (on top)
show.all.cells Show all cells or only query and neighbor cells
... Extra parameters passed to DimPlot

Value
A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

NormalizeData Normalize Data

Description
Normalize the count data present in a given assay.

Usage
NormalizeData(object, ...)

## Default S3 method:
NormalizeData(
object,
normalization.method = "LogNormalize",
scale.factor = 10000,
margin = 1,
block.size = NULL,
verbose = TRUE,
...
)
NormalizeData 133

## S3 method for class 'Assay'

NormalizeData(
object,
normalization.method = "LogNormalize",
scale.factor = 10000,
margin = 1,
verbose = TRUE,
...
)

## S3 method for class 'Seurat'

NormalizeData(
object,
assay = NULL,
normalization.method = "LogNormalize",
scale.factor = 10000,
margin = 1,
verbose = TRUE,
...
)

Arguments

object An object
... Arguments passed to other methods
normalization.method
Method for normalization.
• LogNormalize: Feature counts for each cell are divided by the total counts
for that cell and multiplied by the scale.factor. This is then natural-log
transformed using log1p.
• CLR: Applies a centered log ratio transformation
• RC: Relative counts. Feature counts for each cell are divided by the total
counts for that cell and multiplied by the scale.factor. No log-transformation
is applied. For counts per million (CPM) set scale.factor = 1e6
scale.factor Sets the scale factor for cell-level normalization
margin If performing CLR normalization, normalize across features (1) or cells (2)
block.size How many cells should be run in each chunk, will try to split evenly across
threads
verbose display progress bar for normalization procedure
assay Name of assay to use

Value

Returns object after normalization

134 PCASigGenes

Examples
## Not run:
data("pbmc_small")
pbmc_small
pmbc_small <- NormalizeData(object = pbmc_small)

## End(Not run)

PCASigGenes Significant genes from a PCA

Description
Returns a set of genes, based on the JackStraw analysis, that have statistically significant associa-
tions with a set of PCs.

Usage
PCASigGenes(
object,
pcs.use,
pval.cut = 0.1,
use.full = FALSE,
max.per.pc = NULL
)

Arguments
object Seurat object
pcs.use PCS to use.
pval.cut P-value cutoff
use.full Use the full list of genes (from the projected PCA). Assumes that ProjectDim
has been run. Currently, must be set to FALSE.
max.per.pc Maximum number of genes to return per PC. Used to avoid genes from one PC
dominating the entire analysis.

Value
A vector of genes whose p-values are statistically significant for at least one of the given PCs.

See Also
ProjectDim JackStraw
PercentageFeatureSet 135

Examples
data("pbmc_small")
PCASigGenes(pbmc_small, pcs.use = 1:2)

PercentageFeatureSet Calculate the percentage of all counts that belong to a given set of
features

Description

This function enables you to easily calculate the percentage of all the counts belonging to a subset
of the possible features for each cell. This is useful when trying to compute the percentage of
transcripts that map to mitochondrial genes for example. The calculation here is simply the column
sum of the matrix present in the counts slot for features belonging to the set divided by the column
sum for all features times 100.

Usage

PercentageFeatureSet(
object,
pattern = NULL,
features = NULL,
col.name = NULL,
assay = NULL
)

Arguments

object A Seurat object

pattern A regex pattern to match features against
features A defined feature set. If features provided, will ignore the pattern matching
col.name Name in meta.data column to assign. If this is not null, returns a Seurat object
with the proportion of the feature set stored in metadata.
assay Assay to use

Value

Returns a vector with the proportion of the feature set or if md.name is set, returns a Seurat object
with the proportion of the feature set stored in metadata.
136 PlotClusterTree

Examples

data("pbmc_small")
# Calculate the proportion of transcripts mapping to mitochondrial genes
# NOTE: The pattern provided works for human gene names. You may need to adjust depending on your
# system of interest
pbmc_small[["percent.mt"]] <- PercentageFeatureSet(object = pbmc_small, pattern = "^MT-")

PlotClusterTree Plot clusters as a tree

Description

Plots previously computed tree (from BuildClusterTree)

Usage

PlotClusterTree(object, direction = "downwards", ...)

Arguments

object Seurat object

direction A character string specifying the direction of the tree (default is downwards)
Possible options: "rightwards", "leftwards", "upwards", and "downwards".
... Additional arguments to ape::plot.phylo

Value

Plots dendogram (must be precomputed using BuildClusterTree), returns no value

Examples

if (requireNamespace("ape", quietly = TRUE)) {

data("pbmc_small")
pbmc_small <- BuildClusterTree(object = pbmc_small)
PlotClusterTree(object = pbmc_small)
}
PlotPerturbScore 137

PlotPerturbScore Function to plot perturbation score distributions.

Description
Density plots to visualize perturbation scores calculated from RunMixscape function.

Usage
PlotPerturbScore(
object,
target.gene.class = "gene",
target.gene.ident = NULL,
mixscape.class = "mixscape_class",
col = "orange2",
split.by = NULL,
before.mixscape = FALSE,
prtb.type = "KO"
)

Arguments
object An object of class Seurat.
target.gene.class
meta data column specifying all target gene names in the experiment.
target.gene.ident
Target gene name to visualize perturbation scores for.
mixscape.class meta data column specifying mixscape classifications.
col Specify color of target gene class or knockout cell class. For control non-
targeting and non-perturbed cells, colors are set to different shades of grey.
split.by For datasets with more than one cell type. Set equal TRUE to visualize pertur-
bation scores for each cell type separately.
before.mixscape
Option to split densities based on mixscape classification (default) or original
target gene classification. Default is set to NULL and plots cells by original
class ID.
prtb.type specify type of CRISPR perturbation expected for labeling mixscape classifica-
tions. Default is KO.

Value
A ggplot object.
138 PolyFeaturePlot

PolyDimPlot Polygon DimPlot

Description

Plot cells as polygons, rather than single points. Color cells by identity, or a categorical variable in
metadata

Usage

PolyDimPlot(
object,
group.by = NULL,
cells = NULL,
poly.data = "spatial",
flip.coords = FALSE
)

Arguments

object Seurat object

group.by A grouping variable present in the metadata. Default is to use the groupings
present in the current cell identities (Idents(object = object))
cells Vector of cells to plot (default is all cells)
poly.data Name of the polygon dataframe in the misc slot
flip.coords Flip x and y coordinates

Value

Returns a ggplot object

PolyFeaturePlot Polygon FeaturePlot

Description

Plot cells as polygons, rather than single points. Color cells by any value accessible by FetchData.
PredictAssay 139

Usage
PolyFeaturePlot(
object,
features,
cells = NULL,
poly.data = "spatial",
ncol = ceiling(x = length(x = features)/2),
min.cutoff = 0,
max.cutoff = NA,
common.scale = TRUE,
flip.coords = FALSE
)

Arguments
object Seurat object
features Vector of features to plot. Features can come from:
• An Assay feature (e.g. a gene name - "MS4A1")
• A column name from meta.data (e.g. mitochondrial percentage - "per-
cent.mito")
• A column name from a DimReduc object corresponding to the cell embed-
ding values (e.g. the PC 1 scores - "PC_1")
cells Vector of cells to plot (default is all cells)
poly.data Name of the polygon dataframe in the misc slot
ncol Number of columns to split the plot into
min.cutoff Vector of minimum and maximum cutoff values for each feature, may specify
quantile in the form of ’q##’ where ’##’ is the quantile (eg, ’q1’, ’q10’)
max.cutoff Vector of minimum and maximum cutoff values for each feature, may specify
quantile in the form of ’q##’ where ’##’ is the quantile (eg, ’q1’, ’q10’)
common.scale ...
flip.coords Flip x and y coordinates

Value
Returns a ggplot object

PredictAssay Predict value from nearest neighbors

Description
This function will predict expression or cell embeddings from its k nearest neighbors index. For
each cell, it will average its k neighbors value to get its new imputed value. It can average expression
value in assays and cell embeddings from dimensional reductions.
140 PrepLDA

Usage
PredictAssay(
object,
nn.idx,
assay,
reduction = NULL,
dims = NULL,
return.assay = TRUE,
slot = "scale.data",
features = NULL,
mean.function = rowMeans,
seed = 4273,
verbose = TRUE
)

Arguments
object The object used to calculate knn
nn.idx k near neighbour indices. A cells x k matrix.
assay Assay used for prediction
reduction Cell embedding of the reduction used for prediction
dims Number of dimensions of cell embedding
return.assay Return an assay or a predicted matrix
slot slot used for prediction
features features used for prediction
mean.function the function used to calculate row mean
seed Sets the random seed to check if the nearest neighbor is query cell
verbose Print progress

Value
return an assay containing predicted expression value in the data slot

PrepLDA Function to prepare data for Linear Discriminant Analysis.

Description
This function performs unsupervised PCA on each mixscape class separately and projects each
subspace onto all cells in the data.
PrepSCTIntegration 141

Usage
PrepLDA(
object,
de.assay = "RNA",
pc.assay = "PRTB",
labels = "gene",
nt.label = "NT",
npcs = 10,
verbose = TRUE,
logfc.threshold = 0.25
)

Arguments
object An object of class Seurat.
de.assay Assay to use for selection of DE genes.
pc.assay Assay to use for running Principle components analysis.
labels Meta data column with target gene class labels.
nt.label Name of non-targeting cell class.
npcs Number of principle components to use.
verbose Print progress bar.
logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.

Value
Returns a list of the first 10 PCs from each projection.

PrepSCTIntegration Prepare an object list normalized with sctransform for integration.

Description
This function takes in a list of objects that have been normalized with the SCTransform method and
performs the following steps:
• If anchor.features is a numeric value, calls SelectIntegrationFeatures to determine the
features to use in the downstream integration procedure.
• Ensures that the sctransform residuals for the features specified to anchor.features are present
in each object in the list. This is necessary because the default behavior of SCTransform is to
only store the residuals for the features determined to be variable. Residuals are recomputed
for missing features using the stored model parameters via the GetResidual function.
• Subsets the scale.data slot to only contain the residuals for anchor.features for efficiency in
downstream processing.
142 PrepSCTIntegration

Usage
PrepSCTIntegration(
object.list,
assay = NULL,
anchor.features = 2000,
sct.clip.range = NULL,
verbose = TRUE
)

Arguments
object.list A list of Seurat objects to prepare for integration
assay The name of the Assay to use for integration. This can be a single name if all
the assays to be integrated have the same name, or a character vector containing
the name of each Assay in each object to be integrated. The specified assays
must have been normalized using SCTransform. If NULL (default), the current
default assay for each object is used.
anchor.features
Can be either:
• A numeric value. This will call SelectIntegrationFeatures to select the
provided number of features to be used in anchor finding
• A vector of features to be used as input to the anchor finding process
sct.clip.range Numeric of length two specifying the min and max values the Pearson residual
will be clipped to
verbose Display output/messages

Value
A list of Seurat objects with the appropriate scale.data slots containing only the required anchor.features.

Examples
## Not run:
# to install the SeuratData package see https://github.com/satijalab/seurat-data
library(SeuratData)
data("panc8")

# panc8 is a merged Seurat object containing 8 separate pancreas datasets

# split the object by dataset and take the first 2 to integrate
pancreas.list <- SplitObject(panc8, split.by = "tech")[1:2]

# perform SCTransform normalization

pancreas.list <- lapply(X = pancreas.list, FUN = SCTransform)

# select integration features and prep step

features <- SelectIntegrationFeatures(pancreas.list)
pancreas.list <- PrepSCTIntegration(
pancreas.list,
ProjectDim 143

anchor.features = features
)

# downstream integration steps

anchors <- FindIntegrationAnchors(
pancreas.list,
normalization.method = "SCT",
anchor.features = features
)
pancreas.integrated <- IntegrateData(anchors)

## End(Not run)

ProjectDim Project Dimensional reduction onto full dataset

Description
Takes a pre-computed dimensional reduction (typically calculated on a subset of genes) and projects
this onto the entire dataset (all genes). Note that the cell loadings will remain unchanged, but now
there are gene loadings for all genes.

Usage
ProjectDim(
object,
reduction = "pca",
assay = NULL,
dims.print = 1:5,
nfeatures.print = 20,
overwrite = FALSE,
do.center = FALSE,
verbose = TRUE
)

Arguments
object Seurat object
reduction Reduction to use
assay Assay to use
dims.print Number of dims to print features for
nfeatures.print
Number of features with highest/lowest loadings to print for each dimension
overwrite Replace the existing data in feature.loadings
do.center Center the dataset prior to projection (should be set to TRUE)
verbose Print top genes associated with the projected dimensions
144 ProjectUMAP

Value
Returns Seurat object with the projected values

Examples
data("pbmc_small")
pbmc_small
pbmc_small <- ProjectDim(object = pbmc_small, reduction = "pca")
# Vizualize top projected genes in heatmap
DimHeatmap(object = pbmc_small, reduction = "pca", dims = 1, balanced = TRUE)

ProjectUMAP Project query into UMAP coordinates of a reference

Description
This function will take a query dataset and project it into the coordinates of a provided reference
UMAP. This is essentially a wrapper around two steps:

• FindNeighbors - Find the nearest reference cell neighbors and their distances for each query
cell.
• RunUMAP - Perform umap projection by providing the neighbor set calculated above and the
umap model previously computed in the reference.

Usage
ProjectUMAP(query, ...)

## Default S3 method:
ProjectUMAP(
query,
query.dims = NULL,
reference,
reference.dims = NULL,
k.param = 20,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "cosine",
l2.norm = FALSE,
cache.index = TRUE,
index = NULL,
neighbor.name = "query_ref.nn",
reduction.model,
...
)
ProjectUMAP 145

## S3 method for class 'DimReduc'

ProjectUMAP(
query,
query.dims = NULL,
reference,
reference.dims = NULL,
k.param = 20,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "cosine",
l2.norm = FALSE,
cache.index = TRUE,
index = NULL,
neighbor.name = "query_ref.nn",
reduction.model,
...
)

## S3 method for class 'Seurat'

ProjectUMAP(
query,
query.reduction,
query.dims = NULL,
reference,
reference.reduction,
reference.dims = NULL,
k.param = 20,
nn.method = "annoy",
n.trees = 50,
annoy.metric = "cosine",
l2.norm = FALSE,
cache.index = TRUE,
index = NULL,
neighbor.name = "query_ref.nn",
reduction.model,
reduction.name = "ref.umap",
reduction.key = "refUMAP_",
...
)

Arguments
query Query dataset
... Additional parameters to RunUMAP
query.dims Dimensions (columns) to use from query
reference Reference dataset
reference.dims Dimensions (columns) to use from reference
146 Radius.SlideSeq

k.param Defines k for the k-nearest neighbor algorithm

nn.method Method for nearest neighbor finding. Options include: rann, annoy
n.trees More trees gives higher precision when using annoy approximate nearest neigh-
bor search
annoy.metric Distance metric for annoy. Options include: euclidean, cosine, manhattan, and
hamming
l2.norm Take L2Norm of the data
cache.index Include cached index in returned Neighbor object (only relevant if return.neighbor
= TRUE)
index Precomputed index. Useful if querying new data against existing index to avoid
recomputing.
neighbor.name Name to store neighbor information in the query
reduction.model
DimReduc object that contains the umap model
query.reduction
Name of reduction to use from the query for neighbor finding
reference.reduction
Name of reduction to use from the reference for neighbor finding
reduction.name Name of projected UMAP to store in the query
reduction.key Value for the projected UMAP key

Radius.SlideSeq Get Spot Radius

Description
Get Spot Radius

Usage
## S3 method for class 'SlideSeq'
Radius(object)

## S3 method for class 'STARmap'

Radius(object)

## S3 method for class 'VisiumV1'

Radius(object)

Arguments
object An image object

See Also
SeuratObject::Radius
Read10X 147

Read10X Load in data from 10X

Description
Enables easy loading of sparse data matrices provided by 10X genomics.

Usage
Read10X(
data.dir,
gene.column = 2,
cell.column = 1,
unique.features = TRUE,
strip.suffix = FALSE
)

Arguments
data.dir Directory containing the matrix.mtx, genes.tsv (or features.tsv), and barcodes.tsv
files provided by 10X. A vector or named vector can be given in order to load
several data directories. If a named vector is given, the cell barcode names will
be prefixed with the name.
gene.column Specify which column of genes.tsv or features.tsv to use for gene names; default
is 2
cell.column Specify which column of barcodes.tsv to use for cell names; default is 1
unique.features
Make feature names unique (default TRUE)
strip.suffix Remove trailing "-1" if present in all cell barcodes.

Value
If features.csv indicates the data has multiple data types, a list containing a sparse matrix of the data
from each type will be returned. Otherwise a sparse matrix containing the expression data will be
returned.

Examples
## Not run:
# For output from CellRanger < 3.0
data_dir <- 'path/to/data/directory'
list.files(data_dir) # Should show barcodes.tsv, genes.tsv, and matrix.mtx
expression_matrix <- Read10X(data.dir = data_dir)
seurat_object = CreateSeuratObject(counts = expression_matrix)

# For output from CellRanger >= 3.0 with multiple data types
data_dir <- 'path/to/data/directory'
148 Read10X_Image

list.files(data_dir) # Should show barcodes.tsv.gz, features.tsv.gz, and matrix.mtx.gz

data <- Read10X(data.dir = data_dir)
seurat_object = CreateSeuratObject(counts = data$`Gene Expression`)
seurat_object[['Protein']] = CreateAssayObject(counts = data$`Antibody Capture`)

## End(Not run)

Read10X_h5 Read 10X hdf5 file

Description
Read count matrix from 10X CellRanger hdf5 file. This can be used to read both scATAC-seq and
scRNA-seq matrices.

Usage
Read10X_h5(filename, use.names = TRUE, unique.features = TRUE)

Arguments
filename Path to h5 file
use.names Label row names with feature names rather than ID numbers.
unique.features
Make feature names unique (default TRUE)

Value
Returns a sparse matrix with rows and columns labeled. If multiple genomes are present, returns a
list of sparse matrices (one per genome).

Read10X_Image Load a 10X Genomics Visium Image

Description
Load a 10X Genomics Visium Image

Usage
Read10X_Image(
image.dir,
image.name = "tissue_lowres_image.png",
filter.matrix = TRUE,
...
)
ReadMtx 149

Arguments

image.dir Path to directory with 10X Genomics visium image data; should include files
tissue_lowres_image.png,
image.name The file name of the image. Defaults to tissue_lowres_image.png. scalefactors_json.json
and tissue_positions_list.csv
filter.matrix Filter spot/feature matrix to only include spots that have been determined to be
over tissue.
... Ignored for now

Value

A VisiumV1 object

See Also

VisiumV1 Load10X_Spatial

ReadMtx Load in data from remote or local mtx files

Description

Enables easy loading of sparse data matrices

Usage

ReadMtx(
mtx,
cells,
features,
cell.column = 1,
feature.column = 2,
cell.sep = "\t",
feature.sep = "\t",
skip.cell = 0,
skip.feature = 0,
mtx.transpose = FALSE,
unique.features = TRUE,
strip.suffix = FALSE
)
150 ReadMtx

Arguments

mtx Name or remote URL of the mtx file

cells Name or remote URL of the cells/barcodes file
features Name or remote URL of the features/genes file
cell.column Specify which column of cells file to use for cell names; default is 1
feature.column Specify which column of features files to use for feature/gene names; default is
2
cell.sep Specify the delimiter in the cell name file
feature.sep Specify the delimiter in the feature name file
skip.cell Number of lines to skip in the cells file before beginning to read cell names
skip.feature Number of lines to skip in the features file before beginning to gene names
mtx.transpose Transpose the matrix after reading in
unique.features
Make feature names unique (default TRUE)
strip.suffix Remove trailing "-1" if present in all cell barcodes.

Value

A sparse matrix containing the expression data.

Examples

## Not run:
# For local files:

expression_matrix <- ReadMtx(

mtx = "count_matrix.mtx.gz", features = "features.tsv.gz",
cells = "barcodes.tsv.gz"
)
seurat_object <- CreateSeuratObject(counts = expression_matrix)

# For remote files:

expression_matrix <- ReadMtx(mtx = "http://localhost/matrix.mtx",

cells = "http://localhost/barcodes.tsv",
features = "http://localhost/genes.tsv")
seurat_object <- CreateSeuratObject(counts = data)

## End(Not run)
ReadParseBio 151

ReadParseBio Read output from Parse Biosciences

Description
Read output from Parse Biosciences

Usage
ReadParseBio(data.dir, ...)

Arguments
data.dir Directory containing the data files
... Extra parameters passed to ReadMtx

ReadSlideSeq Load Slide-seq spatial data

Description
Load Slide-seq spatial data

Usage
ReadSlideSeq(coord.file, assay = "Spatial")

Arguments
coord.file Path to csv file containing bead coordinate positions
assay Name of assay to associate image to

Value
A SlideSeq object

See Also
SlideSeq
152 RegroupIdents

ReadSTARsolo Read output from STARsolo

Description
Read output from STARsolo

Usage
ReadSTARsolo(data.dir, ...)

Arguments
data.dir Directory containing the data files
... Extra parameters passed to ReadMtx

RegroupIdents Regroup idents based on meta.data info

Description
For cells in each ident, set a new identity based on the most common value of a specified metadata
column.

Usage
RegroupIdents(object, metadata)

Arguments
object Seurat object
metadata Name of metadata column

Value
A Seurat object with the active idents regrouped

Examples
data("pbmc_small")
pbmc_small <- RegroupIdents(pbmc_small, metadata = "groups")
RelativeCounts 153

RelativeCounts Normalize raw data to fractions

Description
Normalize count data to relative counts per cell by dividing by the total per cell. Optionally use a
scale factor, e.g. for counts per million (CPM) use scale.factor = 1e6.

Usage
RelativeCounts(data, scale.factor = 1, verbose = TRUE)

Arguments
data Matrix with the raw count data
scale.factor Scale the result. Default is 1
verbose Print progress

Value
Returns a matrix with the relative counts

Examples
mat <- matrix(data = rbinom(n = 25, size = 5, prob = 0.2), nrow = 5)
mat
mat_norm <- RelativeCounts(data = mat)
mat_norm

RenameCells.SCTAssay Rename Cells in an Object

Description
Rename Cells in an Object

Usage
## S3 method for class 'SCTAssay'
RenameCells(object, new.names = NULL, ...)

## S3 method for class 'SlideSeq'

RenameCells(object, new.names = NULL, ...)

## S3 method for class 'STARmap'

154 RidgePlot

RenameCells(object, new.names = NULL, ...)

## S3 method for class 'VisiumV1'

RenameCells(object, new.names = NULL, ...)

Arguments

object An object
new.names vector of new cell names
... Arguments passed to other methods

See Also

SeuratObject::RenameCells

RidgePlot Single cell ridge plot

Description

Draws a ridge plot of single cell data (gene expression, metrics, PC scores, etc.)

Usage

RidgePlot(
object,
features,
cols = NULL,
idents = NULL,
sort = FALSE,
assay = NULL,
group.by = NULL,
y.max = NULL,
same.y.lims = FALSE,
log = FALSE,
ncol = NULL,
slot = "data",
stack = FALSE,
combine = TRUE,
fill.by = "feature"
)
RunCCA 155

Arguments

object Seurat object

features Features to plot (gene expression, metrics, PC scores, anything that can be re-
treived by FetchData)
cols Colors to use for plotting
idents Which classes to include in the plot (default is all)
sort Sort identity classes (on the x-axis) by the average expression of the attribute
being potted, can also pass ’increasing’ or ’decreasing’ to change sort direction
assay Name of assay to use, defaults to the active assay
group.by Group (color) cells in different ways (for example, orig.ident)
y.max Maximum y axis value
same.y.lims Set all the y-axis limits to the same values
log plot the feature axis on log scale
ncol Number of columns if multiple plots are displayed
slot Use non-normalized counts data for plotting
stack Horizontally stack plots for each feature
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot
fill.by Color violins/ridges based on either ’feature’ or ’ident’

Value

A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

Examples
data("pbmc_small")
RidgePlot(object = pbmc_small, features = 'PC_1')

RunCCA Perform Canonical Correlation Analysis

Description

Runs a canonical correlation analysis using a diagonal implementation of CCA. For details about
stored CCA calculation parameters, see PrintCCAParams.
156 RunCCA

Usage
RunCCA(object1, object2, ...)

## Default S3 method:
RunCCA(
object1,
object2,
standardize = TRUE,
num.cc = 20,
seed.use = 42,
verbose = FALSE,
...
)

## S3 method for class 'Seurat'

RunCCA(
object1,
object2,
assay1 = NULL,
assay2 = NULL,
num.cc = 20,
features = NULL,
renormalize = FALSE,
rescale = FALSE,
compute.gene.loadings = TRUE,
add.cell.id1 = NULL,
add.cell.id2 = NULL,
verbose = TRUE,
...
)

Arguments
object1 First Seurat object
object2 Second Seurat object.
... Extra parameters (passed onto MergeSeurat in case with two objects passed,
passed onto ScaleData in case with single object and rescale.groups set to TRUE)
standardize Standardize matrices - scales columns to have unit variance and mean 0
num.cc Number of canonical vectors to calculate
seed.use Random seed to set. If NULL, does not set a seed
verbose Show progress messages
assay1, assay2 Assays to pull from in the first and second objects, respectively
features Set of genes to use in CCA. Default is the union of both the variable features
sets present in both objects.
renormalize Renormalize raw data after merging the objects. If FALSE, merge the data ma-
trices also.
RunICA 157

rescale Rescale the datasets prior to CCA. If FALSE, uses existing data in the scale data
slots.
compute.gene.loadings
Also compute the gene loadings. NOTE - this will scale every gene in the dataset
which may impose a high memory cost.
add.cell.id1, add.cell.id2
Add ...

Value
Returns a combined Seurat object with the CCA results stored.

See Also
merge.Seurat

Examples
data("pbmc_small")
pbmc_small
# As CCA requires two datasets, we will split our test object into two just for this example
pbmc1 <- subset(pbmc_small, cells = colnames(pbmc_small)[1:40])
pbmc2 <- subset(pbmc_small, cells = colnames(x = pbmc_small)[41:80])
pbmc1[["group"]] <- "group1"
pbmc2[["group"]] <- "group2"
pbmc_cca <- RunCCA(object1 = pbmc1, object2 = pbmc2)
# Print results
print(x = pbmc_cca[["cca"]])

RunICA Run Independent Component Analysis on gene expression

Description
Run fastica algorithm from the ica package for ICA dimensionality reduction. For details about
stored ICA calculation parameters, see PrintICAParams.

Usage
RunICA(object, ...)

## Default S3 method:
RunICA(
object,
assay = NULL,
nics = 50,
rev.ica = FALSE,
158 RunICA

ica.function = "icafast",
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.name = "ica",
reduction.key = "ica_",
seed.use = 42,
...
)

## S3 method for class 'Assay'

RunICA(
object,
assay = NULL,
features = NULL,
nics = 50,
rev.ica = FALSE,
ica.function = "icafast",
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.name = "ica",
reduction.key = "ica_",
seed.use = 42,
...
)

## S3 method for class 'Seurat'

RunICA(
object,
assay = NULL,
features = NULL,
nics = 50,
rev.ica = FALSE,
ica.function = "icafast",
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.name = "ica",
reduction.key = "IC_",
seed.use = 42,
...
)

Arguments

object Seurat object

... Additional arguments to be passed to fastica
RunLDA 159

assay Name of Assay ICA is being run on

nics Number of ICs to compute
rev.ica By default, computes the dimensional reduction on the cell x feature matrix.
Setting to true will compute it on the transpose (feature x cell matrix).
ica.function ICA function from ica package to run (options: icafast, icaimax, icajade)
verbose Print the top genes associated with high/low loadings for the ICs
ndims.print ICs to print genes for
nfeatures.print
Number of genes to print for each IC
reduction.name dimensional reduction name
reduction.key dimensional reduction key, specifies the string before the number for the dimen-
sion names.
seed.use Set a random seed. Setting NULL will not set a seed.
features Features to compute ICA on

RunLDA Run Linear Discriminant Analysis

Description
Run Linear Discriminant Analysis
Function to perform Linear Discriminant Analysis.

Usage
RunLDA(object, ...)

## Default S3 method:
RunLDA(
object,
labels,
assay = NULL,
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.key = "LDA_",
seed = 42,
...
)

## S3 method for class 'Assay'

RunLDA(
object,
assay = NULL,
160 RunLDA

labels,
features = NULL,
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.key = "LDA_",
seed = 42,
...
)

## S3 method for class 'Seurat'

RunLDA(
object,
assay = NULL,
labels,
features = NULL,
reduction.name = "lda",
reduction.key = "LDA_",
seed = 42,
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
...
)

Arguments

object An object of class Seurat.

... Arguments passed to other methods
labels Meta data column with target gene class labels.
assay Assay to use for performing Linear Discriminant Analysis (LDA).
verbose Print the top genes associated with high/low loadings for the PCs
ndims.print Number of LDA dimensions to print.
nfeatures.print
Number of features to print for each LDA component.
reduction.key Reduction key name.
seed Value for random seed
features Features to compute LDA on
reduction.name dimensional reduction name, lda by default
RunMarkVario 161

RunMarkVario Run the mark variogram computation on a given position matrix and
expression matrix.

Description
Wraps the functionality of markvario from the spatstat package.

Usage
RunMarkVario(spatial.location, data, ...)

Arguments
spatial.location
A 2 column matrix giving the spatial locations of each of the data points also in
data
data Matrix containing the data used as "marks" (e.g. gene expression)
... Arguments passed to markvario

RunMixscape Run Mixscape

Description
Function to identify perturbed and non-perturbed gRNA expressing cells that accounts for multiple
treatments/conditions/chemical perturbations.

Usage
RunMixscape(
object,
assay = "PRTB",
slot = "scale.data",
labels = "gene",
nt.class.name = "NT",
new.class.name = "mixscape_class",
min.de.genes = 5,
min.cells = 5,
de.assay = "RNA",
logfc.threshold = 0.25,
iter.num = 10,
verbose = FALSE,
split.by = NULL,
fine.mode = FALSE,
162 RunMixscape

fine.mode.labels = "guide_ID",
prtb.type = "KO"
)

Arguments
object An object of class Seurat.
assay Assay to use for mixscape classification.
slot Assay data slot to use.
labels metadata column with target gene labels.
nt.class.name Classification name of non-targeting gRNA cells.
new.class.name Name of mixscape classification to be stored in metadata.
min.de.genes Required number of genes that are differentially expressed for method to sepa-
rate perturbed and non-perturbed cells.
min.cells Minimum number of cells in target gene class. If fewer than this many cells are
assigned to a target gene class during classification, all are assigned NP.
de.assay Assay to use when performing differential expression analysis. Usually RNA.
logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-
scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold
speeds up the function, but can miss weaker signals.
iter.num Number of normalmixEM iterations to run if convergence does not occur.
verbose Display messages
split.by metadata column with experimental condition/cell type classification informa-
tion. This is meant to be used to account for cases a perturbation is condition/cell
type -specific.
fine.mode When this is equal to TRUE, DE genes for each target gene class will be calcu-
lated for each gRNA separately and pooled into one DE list for calculating the
perturbation score of every cell and their subsequent classification.
fine.mode.labels
metadata column with gRNA ID labels.
prtb.type specify type of CRISPR perturbation expected for labeling mixscape classifica-
tions. Default is KO.

Value
Returns Seurat object with with the following information in the meta data and tools slots:
mixscape_class Classification result with cells being either classified as perturbed (KO, by default)
or non-perturbed (NP) based on their target gene class.
mixscape_class.global Global classification result (perturbed, NP or NT)
p_ko Posterior probabilities used to determine if a cell is KO (default). Name of this item will
change to match prtb.type parameter setting. (>0.5) or NP
perturbation score Perturbation scores for every cell calculated in the first iteration of the func-
tion.
RunMoransI 163

RunMoransI Compute Moran’s I value.

Description
Wraps the functionality of the Moran.I function from the ape package. Weights are computed as
1/distance.

Usage
RunMoransI(data, pos, verbose = TRUE)

Arguments
data Expression matrix
pos Position matrix
verbose Display messages/progress

RunPCA Run Principal Component Analysis

Description
Run a PCA dimensionality reduction. For details about stored PCA calculation parameters, see
PrintPCAParams.

Usage
RunPCA(object, ...)

## Default S3 method:
RunPCA(
object,
assay = NULL,
npcs = 50,
rev.pca = FALSE,
weight.by.var = TRUE,
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.key = "PC_",
seed.use = 42,
approx = TRUE,
...
)
164 RunPCA

## S3 method for class 'Assay'

RunPCA(
object,
assay = NULL,
features = NULL,
npcs = 50,
rev.pca = FALSE,
weight.by.var = TRUE,
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.key = "PC_",
seed.use = 42,
...
)

## S3 method for class 'Seurat'

RunPCA(
object,
assay = NULL,
features = NULL,
npcs = 50,
rev.pca = FALSE,
weight.by.var = TRUE,
verbose = TRUE,
ndims.print = 1:5,
nfeatures.print = 30,
reduction.name = "pca",
reduction.key = "PC_",
seed.use = 42,
...
)

Arguments
object An object
... Arguments passed to other methods and IRLBA
assay Name of Assay PCA is being run on
npcs Total Number of PCs to compute and store (50 by default)
rev.pca By default computes the PCA on the cell x gene matrix. Setting to true will
compute it on gene x cell matrix.
weight.by.var Weight the cell embeddings by the variance of each PC (weights the gene load-
ings if rev.pca is TRUE)
verbose Print the top genes associated with high/low loadings for the PCs
ndims.print PCs to print genes for
RunSPCA 165

nfeatures.print
Number of genes to print for each PC
reduction.key dimensional reduction key, specifies the string before the number for the dimen-
sion names. PC by default
seed.use Set a random seed. By default, sets the seed to 42. Setting NULL will not set a
seed.
approx Use truncated singular value decomposition to approximate PCA
features Features to compute PCA on. If features=NULL, PCA will be run using the
variable features for the Assay. Note that the features must be present in the
scaled data. Any requested features that are not scaled or have 0 variance will
be dropped, and the PCA will be run using the remaining features.
reduction.name dimensional reduction name, pca by default

Value
Returns Seurat object with the PCA calculation stored in the reductions slot

RunSPCA Run Supervised Principal Component Analysis

Description
Run a supervied PCA (SPCA) dimensionality reduction supervised by a cell-cell kernel. SPCA
is used to capture a linear transformation which maximizes its dependency to the given cell-cell
kernel. We use SNN graph as the kernel to supervise the linear matrix factorization.

Usage
RunSPCA(object, ...)

## Default S3 method:
RunSPCA(
object,
assay = NULL,
npcs = 50,
reduction.key = "SPC_",
graph = NULL,
verbose = FALSE,
seed.use = 42,
...
)

## S3 method for class 'Assay'

RunSPCA(
object,
assay = NULL,
166 RunSPCA

features = NULL,
npcs = 50,
reduction.key = "SPC_",
graph = NULL,
verbose = TRUE,
seed.use = 42,
...
)

## S3 method for class 'Seurat'

RunSPCA(
object,
assay = NULL,
features = NULL,
npcs = 50,
reduction.name = "spca",
reduction.key = "SPC_",
graph = NULL,
verbose = TRUE,
seed.use = 42,
...
)

Arguments
object An object
... Arguments passed to other methods and IRLBA
assay Name of Assay SPCA is being run on
npcs Total Number of SPCs to compute and store (50 by default)
reduction.key dimensional reduction key, specifies the string before the number for the dimen-
sion names. SPC by default
graph Graph used supervised by SPCA
verbose Print the top genes associated with high/low loadings for the SPCs
seed.use Set a random seed. By default, sets the seed to 42. Setting NULL will not set a
seed.
features Features to compute SPCA on. If features=NULL, SPCA will be run using the
variable features for the Assay.
reduction.name dimensional reduction name, spca by default

Value
Returns Seurat object with the SPCA calculation stored in the reductions slot

References
Barshan E, Ghodsi A, Azimifar Z, Jahromi MZ. Supervised principal component analysis: Visual-
ization, classification and regression on subspaces and submanifolds. Pattern Recognition. 2011 Jul
RunTSNE 167

1;44(7):1357-71. https://www.sciencedirect.com/science/article/pii/S0031320310005819?
casa_token=AZMFg5OtPnAAAAAA:_Udu7GJ7G2ed1-XSmr-3IGSISUwcHfMpNtCj-qacXH5SBC4nwzVid36GXI3r8XG8dK5WOQu

RunTSNE Run t-distributed Stochastic Neighbor Embedding

Description
Run t-SNE dimensionality reduction on selected features. Has the option of running in a reduced
dimensional space (i.e. spectral tSNE, recommended), or running based on a set of genes. For
details about stored TSNE calculation parameters, see PrintTSNEParams.

Usage
RunTSNE(object, ...)

## S3 method for class 'matrix'

RunTSNE(
object,
assay = NULL,
seed.use = 1,
tsne.method = "Rtsne",
dim.embed = 2,
reduction.key = "tSNE_",
...
)

## S3 method for class 'DimReduc'

RunTSNE(
object,
cells = NULL,
dims = 1:5,
seed.use = 1,
tsne.method = "Rtsne",
dim.embed = 2,
reduction.key = "tSNE_",
...
)

## S3 method for class 'dist'

RunTSNE(
object,
assay = NULL,
seed.use = 1,
tsne.method = "Rtsne",
dim.embed = 2,
reduction.key = "tSNE_",
168 RunTSNE

...
)

## S3 method for class 'Seurat'

RunTSNE(
object,
reduction = "pca",
cells = NULL,
dims = 1:5,
features = NULL,
seed.use = 1,
tsne.method = "Rtsne",
dim.embed = 2,
distance.matrix = NULL,
reduction.name = "tsne",
reduction.key = "tSNE_",
...
)

Arguments
object Seurat object
... Arguments passed to other methods and to t-SNE call (most commonly used is
perplexity)
assay Name of assay that that t-SNE is being run on
seed.use Random seed for the t-SNE. If NULL, does not set the seed
tsne.method Select the method to use to compute the tSNE. Available methods are:
• Rtsne: Use the Rtsne package Barnes-Hut implementation of tSNE (de-
fault)
• FIt-SNE: Use the FFT-accelerated Interpolation-based t-SNE. Based on
Kluger Lab code found here: https://github.com/KlugerLab/FIt-SNE
dim.embed The dimensional space of the resulting tSNE embedding (default is 2). For
example, set to 3 for a 3d tSNE
reduction.key dimensional reduction key, specifies the string before the number for the dimen-
sion names. tSNE_ by default
cells Which cells to analyze (default, all cells)
dims Which dimensions to use as input features
reduction Which dimensional reduction (e.g. PCA, ICA) to use for the tSNE. Default is
PCA
features If set, run the tSNE on this subset of features (instead of running on a set of
reduced dimensions). Not set (NULL) by default; dims must be NULL to run
on features
distance.matrix
If set, runs tSNE on the given distance matrix instead of data matrix (experimen-
tal)
RunUMAP 169

reduction.name dimensional reduction name, specifies the position in the object$dr list. tsne by
default

RunUMAP Run UMAP

Description
Runs the Uniform Manifold Approximation and Projection (UMAP) dimensional reduction tech-
nique. To run, you must first install the umap-learn python package (e.g. via pip install umap-learn).
Details on this package can be found here: https://github.com/lmcinnes/umap. For a more
in depth discussion of the mathematics underlying UMAP, see the ArXiv paper here: https:
//arxiv.org/abs/1802.03426.

Usage
RunUMAP(object, ...)

## Default S3 method:
RunUMAP(
object,
reduction.key = "UMAP_",
assay = NULL,
reduction.model = NULL,
return.model = FALSE,
umap.method = "uwot",
n.neighbors = 30L,
n.components = 2L,
metric = "cosine",
n.epochs = NULL,
learning.rate = 1,
min.dist = 0.3,
spread = 1,
set.op.mix.ratio = 1,
local.connectivity = 1L,
repulsion.strength = 1,
negative.sample.rate = 5,
a = NULL,
b = NULL,
uwot.sgd = FALSE,
seed.use = 42,
metric.kwds = NULL,
angular.rp.forest = FALSE,
densmap = FALSE,
dens.lambda = 2,
dens.frac = 0.3,
dens.var.shift = 0.1,
170 RunUMAP

verbose = TRUE,
...
)

## S3 method for class 'Graph'

RunUMAP(
object,
assay = NULL,
umap.method = "umap-learn",
n.components = 2L,
metric = "correlation",
n.epochs = 0L,
learning.rate = 1,
min.dist = 0.3,
spread = 1,
repulsion.strength = 1,
negative.sample.rate = 5L,
a = NULL,
b = NULL,
uwot.sgd = FALSE,
seed.use = 42L,
metric.kwds = NULL,
densmap = FALSE,
densmap.kwds = NULL,
verbose = TRUE,
reduction.key = "UMAP_",
...
)

## S3 method for class 'Neighbor'

RunUMAP(object, reduction.model, ...)

## S3 method for class 'Seurat'

RunUMAP(
object,
dims = NULL,
reduction = "pca",
features = NULL,
graph = NULL,
assay = DefaultAssay(object = object),
nn.name = NULL,
slot = "data",
umap.method = "uwot",
reduction.model = NULL,
return.model = FALSE,
n.neighbors = 30L,
n.components = 2L,
metric = "cosine",
RunUMAP 171

n.epochs = NULL,
learning.rate = 1,
min.dist = 0.3,
spread = 1,
set.op.mix.ratio = 1,
local.connectivity = 1L,
repulsion.strength = 1,
negative.sample.rate = 5L,
a = NULL,
b = NULL,
uwot.sgd = FALSE,
seed.use = 42L,
metric.kwds = NULL,
angular.rp.forest = FALSE,
densmap = FALSE,
dens.lambda = 2,
dens.frac = 0.3,
dens.var.shift = 0.1,
verbose = TRUE,
reduction.name = "umap",
reduction.key = "UMAP_",
...
)

Arguments
object An object
... Arguments passed to other methods and UMAP
reduction.key dimensional reduction key, specifies the string before the number for the dimen-
sion names. UMAP by default
assay Assay to pull data for when using features, or assay used to construct Graph
if running UMAP on a Graph
reduction.model
DimReduc object that contains the umap model
return.model whether UMAP will return the uwot model
umap.method UMAP implementation to run. Can be
uwot: Runs umap via the uwot R package
uwot-learn: Runs umap via the uwot R package and return the learned umap
model
umap-learn: Run the Seurat wrapper of the python umap-learn package
n.neighbors This determines the number of neighboring points used in local approximations
of manifold structure. Larger values will result in more global structure being
preserved at the loss of detailed local structure. In general this parameter should
often be in the range 5 to 50.
n.components The dimension of the space to embed into.
172 RunUMAP

metric metric: This determines the choice of metric used to measure distance in the
input space. A wide variety of metrics are already coded, and a user defined
function can be passed as long as it has been JITd by numba.
n.epochs he number of training epochs to be used in optimizing the low dimensional em-
bedding. Larger values result in more accurate embeddings. If NULL is speci-
fied, a value will be selected based on the size of the input dataset (200 for large
datasets, 500 for small).
learning.rate The initial learning rate for the embedding optimization.
min.dist This controls how tightly the embedding is allowed compress points together.
Larger values ensure embedded points are moreevenly distributed, while smaller
values allow the algorithm to optimise more accurately with regard to local
structure. Sensible values are in the range 0.001 to 0.5.
spread The effective scale of embedded points. In combination with min.dist this de-
termines how clustered/clumped the embedded points are.
set.op.mix.ratio
Interpolate between (fuzzy) union and intersection as the set operation used to
combine local fuzzy simplicial sets to obtain a global fuzzy simplicial sets. Both
fuzzy set operations use the product t-norm. The value of this parameter should
be between 0.0 and 1.0; a value of 1.0 will use a pure fuzzy union, while 0.0 will
use a pure fuzzy intersection.
local.connectivity
The local connectivity required - i.e. the number of nearest neighbors that should
be assumed to be connected at a local level. The higher this value the more
connected the manifold becomes locally. In practice this should be not more
than the local intrinsic dimension of the manifold.
repulsion.strength
Weighting applied to negative samples in low dimensional embedding optimiza-
tion. Values higher than one will result in greater weight being given to negative
samples.
negative.sample.rate
The number of negative samples to select per positive sample in the optimization
process. Increasing this value will result in greater repulsive force being applied,
greater optimization cost, but slightly more accuracy.
a More specific parameters controlling the embedding. If NULL, these values are
set automatically as determined by min. dist and spread. Parameter of differen-
tiable approximation of right adjoint functor.
b More specific parameters controlling the embedding. If NULL, these values are
set automatically as determined by min. dist and spread. Parameter of differen-
tiable approximation of right adjoint functor.
uwot.sgd Set uwot::umap(fast_sgd = TRUE); see umap for more details
seed.use Set a random seed. By default, sets the seed to 42. Setting NULL will not set a
seed
metric.kwds A dictionary of arguments to pass on to the metric, such as the p value for
Minkowski distance. If NULL then no arguments are passed on.
RunUMAP 173

angular.rp.forest
Whether to use an angular random projection forest to initialise the approximate
nearest neighbor search. This can be faster, but is mostly on useful for metric
that use an angular style distance such as cosine, correlation etc. In the case of
those metrics angular forests will be chosen automatically.
densmap Whether to use the density-augmented objective of densMAP. Turning on this
option generates an embedding where the local densities are encouraged to be
correlated with those in the original space. Parameters below with the prefix
‘dens’ further control the behavior of this extension. Default is FALSE. Only
compatible with ’umap-learn’ method and version of umap-learn >= 0.5.0
dens.lambda Specific parameter which controls the regularization weight of the density cor-
relation term in densMAP. Higher values prioritize density preservation over the
UMAP objective, and vice versa for values closer to zero. Setting this parameter
to zero is equivalent to running the original UMAP algorithm. Default value is
2.
dens.frac Specific parameter which controls the fraction of epochs (between 0 and 1)
where the density-augmented objective is used in densMAP. The first (1 - dens_frac)
fraction of epochs optimize the original UMAP objective before introducing the
density correlation term. Default is 0.3.
dens.var.shift Specific parameter which specifies a small constant added to the variance of
local radii in the embedding when calculating the density correlation objective
to prevent numerical instability from dividing by a small number. Default is 0.1.
verbose Controls verbosity
densmap.kwds A dictionary of arguments to pass on to the densMAP optimization.
dims Which dimensions to use as input features, used only if features is NULL
reduction Which dimensional reduction (PCA or ICA) to use for the UMAP input. Default
is PCA
features If set, run UMAP on this subset of features (instead of running on a set of re-
duced dimensions). Not set (NULL) by default; dims must be NULL to run on
features
graph Name of graph on which to run UMAP
nn.name Name of knn output on which to run UMAP
slot The slot used to pull data for when using features. data slot is by default.
reduction.name Name to store dimensional reduction under in the Seurat object

Value
Returns a Seurat object containing a UMAP representation

References
McInnes, L, Healy, J, UMAP: Uniform Manifold Approximation and Projection for Dimension
Reduction, ArXiv e-prints 1802.03426, 2018
174 SampleUMI

Examples
## Not run:
data("pbmc_small")
pbmc_small
# Run UMAP map on first 5 PCs
pbmc_small <- RunUMAP(object = pbmc_small, dims = 1:5)
# Plot results
DimPlot(object = pbmc_small, reduction = 'umap')

## End(Not run)

SampleUMI Sample UMI

Description
Downsample each cell to a specified number of UMIs. Includes an option to upsample cells below
specified UMI as well.

Usage
SampleUMI(data, max.umi = 1000, upsample = FALSE, verbose = FALSE)

Arguments
data Matrix with the raw count data
max.umi Number of UMIs to sample to
upsample Upsamples all cells with fewer than max.umi
verbose Display the progress bar

Value
Matrix with downsampled data

Examples
data("pbmc_small")
counts = as.matrix(x = GetAssayData(object = pbmc_small, assay = "RNA", slot = "counts"))
downsampled = SampleUMI(data = counts)
head(x = downsampled)
SaveAnnoyIndex 175

SaveAnnoyIndex Save the Annoy index

Description
Save the Annoy index

Usage
SaveAnnoyIndex(object, file)

Arguments
object A Neighbor object with the annoy index stored
file Path to file to write index to

ScaleData Scale and center the data.

Description
Scales and centers features in the dataset. If variables are provided in vars.to.regress, they are
individually regressed against each feature, and the resulting residuals are then scaled and centered.

Usage
ScaleData(object, ...)

## Default S3 method:
ScaleData(
object,
features = NULL,
vars.to.regress = NULL,
latent.data = NULL,
split.by = NULL,
model.use = "linear",
use.umi = FALSE,
do.scale = TRUE,
do.center = TRUE,
scale.max = 10,
block.size = 1000,
min.cells.to.block = 3000,
verbose = TRUE,
...
)
176 ScaleData

## S3 method for class 'Assay'

ScaleData(
object,
features = NULL,
vars.to.regress = NULL,
latent.data = NULL,
split.by = NULL,
model.use = "linear",
use.umi = FALSE,
do.scale = TRUE,
do.center = TRUE,
scale.max = 10,
block.size = 1000,
min.cells.to.block = 3000,
verbose = TRUE,
...
)

## S3 method for class 'Seurat'

ScaleData(
object,
features = NULL,
assay = NULL,
vars.to.regress = NULL,
split.by = NULL,
model.use = "linear",
use.umi = FALSE,
do.scale = TRUE,
do.center = TRUE,
scale.max = 10,
block.size = 1000,
min.cells.to.block = 3000,
verbose = TRUE,
...
)

Arguments
object An object
... Arguments passed to other methods
features Vector of features names to scale/center. Default is variable features.
vars.to.regress
Variables to regress out (previously latent.vars in RegressOut). For example,
nUMI, or percent.mito.
latent.data Extra data to regress out, should be cells x latent data
split.by Name of variable in object metadata or a vector or factor defining grouping of
cells. See argument f in split for more details
ScaleFactors 177

model.use Use a linear model or generalized linear model (poisson, negative binomial) for
the regression. Options are ’linear’ (default), ’poisson’, and ’negbinom’
use.umi Regress on UMI count data. Default is FALSE for linear modeling, but auto-
matically set to TRUE if model.use is ’negbinom’ or ’poisson’
do.scale Whether to scale the data.
do.center Whether to center the data.
scale.max Max value to return for scaled data. The default is 10. Setting this can help
reduce the effects of features that are only expressed in a very small number of
cells. If regressing out latent variables and using a non-linear model, the default
is 50.
block.size Default size for number of features to scale at in a single computation. Increas-
ing block.size may speed up calculations but at an additional memory cost.
min.cells.to.block
If object contains fewer than this number of cells, don’t block for scaling calcu-
lations.
verbose Displays a progress bar for scaling procedure
assay Name of Assay to scale

Details
ScaleData now incorporates the functionality of the function formerly known as RegressOut (which
regressed out given the effects of provided variables and then scaled the residuals). To make use
of the regression functionality, simply pass the variables you want to remove to the vars.to.regress
parameter.
Setting center to TRUE will center the expression for each feature by subtracting the average ex-
pression for that feature. Setting scale to TRUE will scale the expression level for each feature by
dividing the centered feature expression levels by their standard deviations if center is TRUE and
by their root mean square otherwise.

ScaleFactors Get image scale factors

Description
Get image scale factors

Usage
ScaleFactors(object, ...)

scalefactors(spot, fiducial, hires, lowres)

## S3 method for class 'VisiumV1'

ScaleFactors(object, ...)

## S3 method for class 'VisiumV1'

ScaleFactors(object, ...)
178 ScoreJackStraw

Arguments
object An object to get scale factors from
... Arguments passed to other methods
spot Spot full resolution scale factor
fiducial Fiducial full resolution scale factor
hires High resolutoin scale factor
lowres Low resolution scale factor

Value
An object of class scalefactors

Note
scalefactors objects can be created with scalefactors()

ScoreJackStraw Compute Jackstraw scores significance.

Description
Significant PCs should show a p-value distribution that is strongly skewed to the left compared to
the null distribution. The p-value for each PC is based on a proportion test comparing the number
of features with a p-value below a particular threshold (score.thresh), compared with the proportion
of features expected under a uniform distribution of p-values.

Usage
ScoreJackStraw(object, ...)

## S3 method for class 'JackStrawData'

ScoreJackStraw(object, dims = 1:5, score.thresh = 1e-05, ...)

## S3 method for class 'DimReduc'

ScoreJackStraw(object, dims = 1:5, score.thresh = 1e-05, ...)

## S3 method for class 'Seurat'

ScoreJackStraw(
object,
reduction = "pca",
dims = 1:5,
score.thresh = 1e-05,
do.plot = FALSE,
...
)
SCTAssay-class 179

Arguments
object An object
... Arguments passed to other methods
dims Which dimensions to examine
score.thresh Threshold to use for the proportion test of PC significance (see Details)
reduction Reduction associated with JackStraw to score
do.plot Show plot. To return ggplot object, use JackStrawPlot after running Score-
JackStraw.

Value
Returns a Seurat object

Author(s)
Omri Wurtzel

See Also
JackStrawPlot
JackStrawPlot

SCTAssay-class The SCTModel Class

Description
The SCTModel object is a model and parameters storage from SCTransform. It can be used to
calculate Pearson residuals for new genes.
The SCTAssay object contains all the information found in an Assay object, with extra information
from the results of SCTransform

Usage
## S3 method for class 'SCTAssay'
levels(x)

## S3 replacement method for class 'SCTAssay'

levels(x) <- value

Arguments
x An SCTAssay object
value New levels, must be in the same order as the levels present
180 SCTAssay-class

Value
levels: SCT model names
levels<-: x with updated SCT model names

Slots
feature.attributes A data.frame with feature attributes in SCTransform
cell.attributes A data.frame with cell attributes in SCTransform
clips A list of two numeric of length two specifying the min and max values the Pearson residual
will be clipped to. One for vst and one for SCTransform
umi.assay Name of the assay of the seurat object containing UMI matrix and the default is RNA
model A formula used in SCTransform
arguments other information used in SCTransform
SCTModel.list A list containing SCT models

Get and set SCT model names

SCT results are named by initial run of SCTransform in order to keep SCT parameters straight
between runs. When working with merged SCTAssay objects, these model names are impor-
tant. levels allows querying the models present. levels<- allows the changing of the names
of the models present, useful when merging SCTAssay objects. Note: unlike normal levels<-,
levels<-.SCTAssay allows complete changing of model names, not reordering.

Creating an SCTAssay from an Assay

Conversion from an Assay object to an SCTAssay object by is done by adding the additional slots
to the object. If from has results generated by SCTransform from Seurat v3.0.0 to v3.1.1, the
conversion will automagically fill the new slots with the data

See Also
Assay
Assay

Examples
## Not run:
# SCTAssay objects are generated from SCTransform
pbmc_small <- SCTransform(pbmc_small)

## End(Not run)

# SCTAssay objects are generated from SCTransform

pbmc_small <- SCTransform(pbmc_small)
pbmc_small[["SCT"]]

## Not run:
SCTransform 181

# Query and change SCT model names

levels(pbmc_small[['SCT']])
levels(pbmc_small[['SCT']]) <- '3'
levels(pbmc_small[['SCT']])

## End(Not run)

SCTransform Use regularized negative binomial regression to normalize UMI count

data

Description
This function calls sctransform::vst. The sctransform package is available at https://github.com/ChristophH/sctransform.
Use this function as an alternative to the NormalizeData, FindVariableFeatures, ScaleData work-
flow. Results are saved in a new assay (named SCT by default) with counts being (corrected)
counts, data being log1p(counts), scale.data being pearson residuals; sctransform::vst intermediate
results are saved in misc slot of new assay.

Usage
SCTransform(
object,
assay = "RNA",
new.assay.name = "SCT",
reference.SCT.model = NULL,
do.correct.umi = TRUE,
ncells = 5000,
residual.features = NULL,
variable.features.n = 3000,
variable.features.rv.th = 1.3,
vars.to.regress = NULL,
do.scale = FALSE,
do.center = TRUE,
clip.range = c(-sqrt(x = ncol(x = object[[assay]])/30), sqrt(x = ncol(x =
object[[assay]])/30)),
conserve.memory = FALSE,
return.only.var.genes = TRUE,
seed.use = 1448145,
verbose = TRUE,
...
)

Arguments
object A seurat object
assay Name of assay to pull the count data from; default is ’RNA’
182 SCTransform

new.assay.name Name for the new assay containing the normalized data
reference.SCT.model
If not NULL, compute residuals for the object using the provided SCT model;
supports only log_umi as the latent variable. If residual.features are not spec-
ified, compute for the top variable.features.n specified in the model which are
also present in the object. If residual.features are specified, the variable features
of the resulting SCT assay are set to the top variable.features.n in the model.
do.correct.umi Place corrected UMI matrix in assay counts slot; default is TRUE
ncells Number of subsampling cells used to build NB regression; default is 5000
residual.features
Genes to calculate residual features for; default is NULL (all genes). If specified,
will be set to VariableFeatures of the returned object.
variable.features.n
Use this many features as variable features after ranking by residual variance;
default is 3000. Only applied if residual.features is not set.
variable.features.rv.th
Instead of setting a fixed number of variable features, use this residual variance
cutoff; this is only used when variable.features.n is set to NULL; default is
1.3. Only applied if residual.features is not set.
vars.to.regress
Variables to regress out in a second non-regularized linear regression. For ex-
ample, percent.mito. Default is NULL
do.scale Whether to scale residuals to have unit variance; default is FALSE
do.center Whether to center residuals to have mean zero; default is TRUE
clip.range Range to clip the residuals to; default is c(-sqrt(n/30),sqrt(n/30)), where
n is the number of cells
conserve.memory
If set to TRUE the residual matrix for all genes is never created in full; useful for
large data sets, but will take longer to run; this will also set return.only.var.genes
to TRUE; default is FALSE
return.only.var.genes
If set to TRUE the scale.data matrices in output assay are subset to contain only
the variable genes; default is TRUE
seed.use Set a random seed. By default, sets the seed to 1448145. Setting NULL will not
set a seed.
verbose Whether to print messages and progress bars
... Additional parameters passed to sctransform::vst

Value
Returns a Seurat object with a new assay (named SCT by default) with counts being (corrected)
counts, data being log1p(counts), scale.data being pearson residuals; sctransform::vst intermediate
results are saved in misc slot of the new assay.

See Also
correct_counts get_residuals
SCTResults 183

Examples
data("pbmc_small")
SCTransform(object = pbmc_small)

SCTResults Get SCT results from an Assay

Description

Pull the SCTResults information from an SCTAssay object.

Usage

SCTResults(object, ...)

SCTResults(object, ...) <- value

## S3 method for class 'SCTModel'

SCTResults(object, slot, ...)

## S3 replacement method for class 'SCTModel'

SCTResults(object, slot, ...) <- value

## S3 method for class 'SCTAssay'

SCTResults(object, slot, model = NULL, ...)

## S3 replacement method for class 'SCTAssay'

SCTResults(object, slot, model = NULL, ...) <- value

## S3 method for class 'Seurat'

SCTResults(object, assay = "SCT", slot, model = NULL, ...)

Arguments

object An object
... Arguments passed to other methods (not used)
value new data to set
slot Which slot to pull the SCT results from
model Name of SCModel to pull result from. Available names can be retrieved with
levels.
assay Assay in the Seurat object to pull from
184 SelectIntegrationFeatures

Value
Returns the value present in the requested slot for the requested group. If group is not specified,
returns a list of slot results for each group unless there is only one group present (in which case it
just returns the slot directly).

SelectIntegrationFeatures
Select integration features

Description
Choose the features to use when integrating multiple datasets. This function ranks features by the
number of datasets they are deemed variable in, breaking ties by the median variable feature rank
across datasets. It returns the top scoring features by this ranking.

Usage
SelectIntegrationFeatures(
object.list,
nfeatures = 2000,
assay = NULL,
verbose = TRUE,
fvf.nfeatures = 2000,
...
)

Arguments
object.list List of seurat objects
nfeatures Number of features to return
assay Name or vector of assay names (one for each object) from which to pull the
variable features.
verbose Print messages
fvf.nfeatures nfeatures for FindVariableFeatures. Used if VariableFeatures have not
been set for any object in object.list.
... Additional parameters to FindVariableFeatures

Details
If for any assay in the list, FindVariableFeatures hasn’t been run, this method will try to run it
using the fvf.nfeatures parameter and any additional ones specified through the . . . .

Value
A vector of selected features
SetIntegrationData 185

Examples
## Not run:
# to install the SeuratData package see https://github.com/satijalab/seurat-data
library(SeuratData)
data("panc8")

# panc8 is a merged Seurat object containing 8 separate pancreas datasets

# split the object by dataset and take the first 2
pancreas.list <- SplitObject(panc8, split.by = "tech")[1:2]

# perform SCTransform normalization

pancreas.list <- lapply(X = pancreas.list, FUN = SCTransform)

# select integration features

features <- SelectIntegrationFeatures(pancreas.list)

## End(Not run)

SetIntegrationData Set integration data

Description

Set integration data

Usage

SetIntegrationData(object, integration.name, slot, new.data)

Arguments

object Seurat object

integration.name
Name of integration object
slot Which slot in integration object to set
new.data New data to insert

Value

Returns a Seurat object

186 SeuratTheme

Seurat-class The Seurat Class

Description
The Seurat object is a representation of single-cell expression data for R; for more details, please
see the documentation in SeuratObject

See Also
SeuratObject::Seurat-class

SeuratCommand-class The SeuratCommand Class

Description
For more details, please see the documentation in SeuratObject

See Also
SeuratObject::SeuratCommand-class

SeuratTheme Seurat Themes

Description
Various themes to be applied to ggplot2-based plots
SeuratTheme The curated Seurat theme, consists of ...
DarkTheme A dark theme, axes and text turn to white, the background becomes black
NoAxes Removes axis lines, text, and ticks
NoLegend Removes the legend
FontSize Sets axis and title font sizes
NoGrid Removes grid lines
SeuratAxes Set Seurat-style axes
SpatialTheme A theme designed for spatial visualizations (eg PolyFeaturePlot, PolyDimPlot)
RestoreLegend Restore a legend after removal
RotatedAxis Rotate X axis text 45 degrees
BoldTitle Enlarges and emphasizes the title
SeuratTheme 187

Usage
SeuratTheme()

CenterTitle(...)

DarkTheme(...)

FontSize(
x.text = NULL,
y.text = NULL,
x.title = NULL,
y.title = NULL,
main = NULL,
...
)

NoAxes(..., keep.text = FALSE, keep.ticks = FALSE)

NoLegend(...)

NoGrid(...)

SeuratAxes(...)

SpatialTheme(...)

RestoreLegend(..., position = "right")

RotatedAxis(...)

BoldTitle(...)

WhiteBackground(...)

Arguments
... Extra parameters to be passed to theme
x.text, y.text X and Y axis text sizes
x.title, y.title
X and Y axis title sizes
main Plot title size
keep.text Keep axis text
keep.ticks Keep axis ticks
position A position to restore the legend to

Value
A ggplot2 theme object
188 SlideSeq-class

See Also
theme

Examples
# Generate a plot with a dark theme
library(ggplot2)
df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
p <- ggplot(data = df, mapping = aes(x = x, y = y)) + geom_point(mapping = aes(color = 'red'))
p + DarkTheme(legend.position = 'none')

# Generate a plot with no axes

library(ggplot2)
df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
p <- ggplot(data = df, mapping = aes(x = x, y = y)) + geom_point(mapping = aes(color = 'red'))
p + NoAxes()

# Generate a plot with no legend

library(ggplot2)
df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
p <- ggplot(data = df, mapping = aes(x = x, y = y)) + geom_point(mapping = aes(color = 'red'))
p + NoLegend()

# Generate a plot with no grid lines

library(ggplot2)
df <- data.frame(x = rnorm(n = 100, mean = 20, sd = 2), y = rbinom(n = 100, size = 100, prob = 0.2))
p <- ggplot(data = df, mapping = aes(x = x, y = y)) + geom_point(mapping = aes(color = 'red'))
p + NoGrid()

SlideSeq-class The SlideSeq class

Description
The SlideSeq class represents spatial information from the Slide-seq platform

Slots
coordinates ...

Slots
assay Name of assay to associate image data with; will give this image priority for visualization
when the assay is set as the active/default assay in a Seurat object
key Key for the image
SpatialImage-class 189

SpatialImage-class The SpatialImage Class

Description
For more details, please see the documentation in SeuratObject

See Also
SeuratObject::SpatialImage-class

SpatialPlot Visualize spatial clustering and expression data.

Description
SpatialPlot plots a feature or discrete grouping (e.g. cluster assignments) as spots over the image
that was collected. We also provide SpatialFeaturePlot and SpatialDimPlot as wrapper functions
around SpatialPlot for a consistent naming framework.

Usage
SpatialPlot(
object,
group.by = NULL,
features = NULL,
images = NULL,
cols = NULL,
image.alpha = 1,
crop = TRUE,
slot = "data",
min.cutoff = NA,
max.cutoff = NA,
cells.highlight = NULL,
cols.highlight = c("#DE2D26", "grey50"),
facet.highlight = FALSE,
label = FALSE,
label.size = 5,
label.color = "white",
label.box = TRUE,
repel = FALSE,
ncol = NULL,
combine = TRUE,
pt.size.factor = 1.6,
alpha = c(1, 1),
190 SpatialPlot

stroke = 0.25,
interactive = FALSE,
do.identify = FALSE,
identify.ident = NULL,
do.hover = FALSE,
information = NULL
)

SpatialDimPlot(
object,
group.by = NULL,
images = NULL,
cols = NULL,
crop = TRUE,
cells.highlight = NULL,
cols.highlight = c("#DE2D26", "grey50"),
facet.highlight = FALSE,
label = FALSE,
label.size = 7,
label.color = "white",
repel = FALSE,
ncol = NULL,
combine = TRUE,
pt.size.factor = 1.6,
alpha = c(1, 1),
image.alpha = 1,
stroke = 0.25,
label.box = TRUE,
interactive = FALSE,
information = NULL
)

SpatialFeaturePlot(
object,
features,
images = NULL,
crop = TRUE,
slot = "data",
min.cutoff = NA,
max.cutoff = NA,
ncol = NULL,
combine = TRUE,
pt.size.factor = 1.6,
alpha = c(1, 1),
image.alpha = 1,
stroke = 0.25,
interactive = FALSE,
information = NULL
SpatialPlot 191

Arguments
object A Seurat object
group.by Name of meta.data column to group the data by
features Name of the feature to visualize. Provide either group.by OR features, not both.
images Name of the images to use in the plot(s)
cols Vector of colors, each color corresponds to an identity class. This may also be
a single character or numeric value corresponding to a palette as specified by
brewer.pal.info. By default, ggplot2 assigns colors
image.alpha Adjust the opacity of the background images. Set to 0 to remove.
crop Crop the plot in to focus on points plotted. Set to FALSE to show entire back-
ground image.
slot If plotting a feature, which data slot to pull from (counts, data, or scale.data)
min.cutoff, max.cutoff
Vector of minimum and maximum cutoff values for each feature, may specify
quantile in the form of ’q##’ where ’##’ is the quantile (eg, ’q1’, ’q10’)
cells.highlight
A list of character or numeric vectors of cells to highlight. If only one group of
cells desired, can simply pass a vector instead of a list. If set, colors selected
cells to the color(s) in cols.highlight
cols.highlight A vector of colors to highlight the cells as; ordered the same as the groups in
cells.highlight; last color corresponds to unselected cells.
facet.highlight
When highlighting certain groups of cells, split each group into its own plot
label Whether to label the clusters
label.size Sets the size of the labels
label.color Sets the color of the label text
label.box Whether to put a box around the label text (geom_text vs geom_label)
repel Repels the labels to prevent overlap
ncol Number of columns if plotting multiple plots
combine Combine plots into a single gg object; note that if TRUE; themeing will not
work when plotting multiple features/groupings
pt.size.factor Scale the size of the spots.
alpha Controls opacity of spots. Provide as a vector specifying the min and max for
SpatialFeaturePlot. For SpatialDimPlot, provide a single alpha value for each
plot.
stroke Control the width of the border around the spots
interactive Launch an interactive SpatialDimPlot or SpatialFeaturePlot session, see ISpatialDimPlot
or ISpatialFeaturePlot for more details
do.identify, do.hover
DEPRECATED in favor of interactive
identify.ident DEPRECATED
information An optional dataframe or matrix of extra information to be displayed on hover
192 SplitObject

Value

If do.identify, either a vector of cells selected or the object with selected cells set to the value of
identify.ident (if set). Else, if do.hover, a plotly object with interactive graphics. Else, a ggplot
object

Examples

## Not run:
# For functionality analagous to FeaturePlot
SpatialPlot(seurat.object, features = "MS4A1")
SpatialFeaturePlot(seurat.object, features = "MS4A1")

# For functionality analagous to DimPlot

SpatialPlot(seurat.object, group.by = "clusters")
SpatialDimPlot(seurat.object, group.by = "clusters")

## End(Not run)

SplitObject Splits object into a list of subsetted objects.

Description

Splits object based on a single attribute into a list of subsetted objects, one for each level of the
attribute. For example, useful for taking an object that contains cells from many patients, and
subdividing it into patient-specific objects.

Usage

SplitObject(object, split.by = "ident")

Arguments

object Seurat object

split.by Attribute for splitting. Default is "ident". Currently only supported for class-
level (i.e. non-quantitative) attributes.

Value

A named list of Seurat objects, each containing a subset of cells from the original object.
STARmap-class 193

Examples
data("pbmc_small")
# Assign the test object a three level attribute
groups <- sample(c("group1", "group2", "group3"), size = 80, replace = TRUE)
names(groups) <- colnames(pbmc_small)
pbmc_small <- AddMetaData(object = pbmc_small, metadata = groups, col.name = "group")
obj.list <- SplitObject(pbmc_small, split.by = "group")

STARmap-class The STARmap class

Description
The STARmap class

Slots
assay Name of assay to associate image data with; will give this image priority for visualization
when the assay is set as the active/default assay in a Seurat object
key Key for the image

subset.AnchorSet Subset an AnchorSet object

Description
Subset an AnchorSet object

Usage
## S3 method for class 'AnchorSet'
subset(
x,
score.threshold = NULL,
disallowed.dataset.pairs = NULL,
dataset.matrix = NULL,
group.by = NULL,
disallowed.ident.pairs = NULL,
ident.matrix = NULL,
...
)
194 SubsetByBarcodeInflections

Arguments
x object to be subsetted.
score.threshold
Only anchor pairs with scores greater than this value are retained.
disallowed.dataset.pairs
Remove any anchors formed between the provided pairs. E.g. list(c(1,5),c(1,2))
filters out any anchors between datasets 1 and 5 and datasets 1 and 2.
dataset.matrix Provide a binary matrix specifying whether a dataset pair is allowable (1) or not
(0). Should be a dataset x dataset matrix.
group.by Grouping variable to determine allowable ident pairs
disallowed.ident.pairs
Remove any anchors formed between provided ident pairs. E.g. list(c("CD4","CD8"),c("B-cell","T-
ident.matrix Provide a binary matrix specifying whether an ident pair is allowable (1) or not
(0). Should be an ident x ident symmetric matrix
... further arguments to be passed to or from other methods.

Value
Returns an AnchorSet object with specified anchors filtered out

SubsetByBarcodeInflections
Subset a Seurat Object based on the Barcode Distribution Inflection
Points

Description
This convenience function subsets a Seurat object based on calculated inflection points.

Usage
SubsetByBarcodeInflections(object)

Arguments
object Seurat object

Details
See [CalculateBarcodeInflections()] to calculate inflection points and [BarcodeInflectionsPlot()] to
visualize and test inflection point calculations.

Value
Returns a subsetted Seurat object.
TopCells 195

Author(s)

Robert A. Amezquita, <[email protected]>

See Also

CalculateBarcodeInflections BarcodeInflectionsPlot

Examples
data("pbmc_small")
pbmc_small <- CalculateBarcodeInflections(
object = pbmc_small,
group.column = 'groups',
threshold.low = 20,
threshold.high = 30
)
SubsetByBarcodeInflections(object = pbmc_small)

TopCells Find cells with highest scores for a given dimensional reduction tech-
nique

Description

Return a list of genes with the strongest contribution to a set of components

Usage

TopCells(object, dim = 1, ncells = 20, balanced = FALSE, ...)

Arguments

object DimReduc object

dim Dimension to use
ncells Number of cells to return
balanced Return an equal number of cells with both + and - scores.
... Extra parameters passed to Embeddings

Value

Returns a vector of cells

196 TopFeatures

Examples
data("pbmc_small")
pbmc_small
head(TopCells(object = pbmc_small[["pca"]]))
# Can specify which dimension and how many cells to return
TopCells(object = pbmc_small[["pca"]], dim = 2, ncells = 5)

TopFeatures Find features with highest scores for a given dimensional reduction
technique

Description
Return a list of features with the strongest contribution to a set of components

Usage
TopFeatures(
object,
dim = 1,
nfeatures = 20,
projected = FALSE,
balanced = FALSE,
...
)

Arguments
object DimReduc object
dim Dimension to use
nfeatures Number of features to return
projected Use the projected feature loadings
balanced Return an equal number of features with both + and - scores.
... Extra parameters passed to Loadings

Value
Returns a vector of features

Examples
data("pbmc_small")
pbmc_small
TopFeatures(object = pbmc_small[["pca"]], dim = 1)
# After projection:
TopFeatures(object = pbmc_small[["pca"]], dim = 1, projected = TRUE)
TopNeighbors 197

TopNeighbors Get nearest neighbors for given cell

Description
Return a vector of cell names of the nearest n cells.

Usage
TopNeighbors(object, cell, n = 5)

Arguments
object Neighbor object
cell Cell of interest
n Number of neighbors to return

Value
Returns a vector of cell names

TransferAnchorSet-class
The TransferAnchorSet Class

Description
Inherits from the Anchorset class. Implemented mainly for method dispatch purposes. See AnchorSet
for slot details.

TransferData Transfer data

Description
Transfer categorical or continuous data across single-cell datasets. For transferring categorical in-
formation, pass a vector from the reference dataset (e.g. refdata = reference$celltype). For
transferring continuous information, pass a matrix from the reference dataset (e.g. refdata =
GetAssayData(reference[['RNA']])).
198 TransferData

Usage
TransferData(
anchorset,
refdata,
reference = NULL,
query = NULL,
weight.reduction = "pcaproject",
l2.norm = FALSE,
dims = NULL,
k.weight = 50,
sd.weight = 1,
eps = 0,
n.trees = 50,
verbose = TRUE,
slot = "data",
prediction.assay = FALSE,
store.weights = TRUE
)

Arguments
anchorset An AnchorSet object generated by FindTransferAnchors
refdata Data to transfer. This can be specified in one of two ways:
• The reference data itself as either a vector where the names correspond to
the reference cells, or a matrix, where the column names correspond to the
reference cells.
• The name of the metadata field or assay from the reference object provided.
This requires the reference parameter to be specified. If pulling assay data
in this manner, it will pull the data from the data slot. To transfer data from
other slots, please pull the data explicitly with GetAssayData and provide
that matrix here.
reference Reference object from which to pull data to transfer
query Query object into which the data will be transferred.
weight.reduction
Dimensional reduction to use for the weighting anchors. Options are:
• pcaproject: Use the projected PCA used for anchor building
• lsiproject: Use the projected LSI used for anchor building
• pca: Use an internal PCA on the query only
• cca: Use the CCA used for anchor building
• custom DimReduc: User provided DimReduc object computed on the query
cells
l2.norm Perform L2 normalization on the cell embeddings after dimensional reduction
dims Set of dimensions to use in the anchor weighting procedure. If NULL, the same
dimensions that were used to find anchors will be used for weighting.
k.weight Number of neighbors to consider when weighting anchors
TransferData 199

sd.weight Controls the bandwidth of the Gaussian kernel for weighting

eps Error bound on the neighbor finding algorithm (from RANN)
n.trees More trees gives higher precision when using annoy approximate nearest neigh-
bor search
verbose Print progress bars and output
slot Slot to store the imputed data. Must be either "data" (default) or "counts"
prediction.assay
Return an Assay object with the prediction scores for each class stored in the
data slot.
store.weights Optionally store the weights matrix used for predictions in the returned query
object.

Details
The main steps of this procedure are outlined below. For a more detailed description of the method-
ology, please see Stuart, Butler, et al Cell 2019. doi: 10.1016/j.cell.2019.05.031; doi: 10.1101/
460147
For both transferring discrete labels and also feature imputation, we first compute the weights ma-
trix.
• Construct a weights matrix that defines the association between each query cell and each
anchor. These weights are computed as 1 - the distance between the query cell and the anchor
divided by the distance of the query cell to the k.weightth anchor multiplied by the anchor
score computed in FindIntegrationAnchors. We then apply a Gaussian kernel width a
bandwidth defined by sd.weight and normalize across all k.weight anchors.
The main difference between label transfer (classification) and feature imputation is what gets mul-
tiplied by the weights matrix. For label transfer, we perform the following steps:
• Create a binary classification matrix, the rows corresponding to each possible class and the
columns corresponding to the anchors. If the reference cell in the anchor pair is a member of
a certain class, that matrix entry is filled with a 1, otherwise 0.
• Multiply this classification matrix by the transpose of weights matrix to compute a prediction
score for each class for each cell in the query dataset.
For feature imputation, we perform the following step:
• Multiply the expression matrix for the reference anchor cells by the weights matrix. This re-
turns a predicted expression matrix for the specified features for each cell in the query dataset.

Value
If query is not provided, for the categorical data in refdata, returns a data.frame with label pre-
dictions. If refdata is a matrix, returns an Assay object where the imputed data has been stored in
the provided slot.
If query is provided, a modified query object is returned. For the categorical data in refdata, pre-
diction scores are stored as Assays (prediction.score.NAME) and two additional metadata fields:
predicted.NAME and predicted.NAME.score which contain the class prediction and the score for
that predicted class. For continuous data, an Assay called NAME is returned. NAME here corre-
sponds to the name of the element in the refdata list.
200 UpdateSCTAssays

References
Stuart T, Butler A, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019;177:1888-1902
doi: 10.1016/j.cell.2019.05.031

Examples
## Not run:
# to install the SeuratData package see https://github.com/satijalab/seurat-data
library(SeuratData)
data("pbmc3k")

# for demonstration, split the object into reference and query

pbmc.reference <- pbmc3k[, 1:1350]
pbmc.query <- pbmc3k[, 1351:2700]

# perform standard preprocessing on each object

pbmc.reference <- NormalizeData(pbmc.reference)
pbmc.reference <- FindVariableFeatures(pbmc.reference)
pbmc.reference <- ScaleData(pbmc.reference)

pbmc.query <- NormalizeData(pbmc.query)

pbmc.query <- FindVariableFeatures(pbmc.query)
pbmc.query <- ScaleData(pbmc.query)

# find anchors
anchors <- FindTransferAnchors(reference = pbmc.reference, query = pbmc.query)

# transfer labels
predictions <- TransferData(anchorset = anchors, refdata = pbmc.reference$seurat_annotations)
pbmc.query <- AddMetaData(object = pbmc.query, metadata = predictions)

## End(Not run)

UpdateSCTAssays Update pre-V4 Assays generated with SCTransform in the Seurat to

the new SCTAssay class

Description
Update pre-V4 Assays generated with SCTransform in the Seurat to the new SCTAssay class

Usage
UpdateSCTAssays(object)

Arguments
object A Seurat object
UpdateSymbolList 201

Value
A Seurat object with updated SCTAssays

UpdateSymbolList Get updated synonyms for gene symbols

Description
Find current gene symbols based on old or alias symbols using the gene names database from the
HUGO Gene Nomenclature Committee (HGNC)

Usage
GeneSymbolThesarus(
symbols,
timeout = 10,
several.ok = FALSE,
search.types = c("alias_symbol", "prev_symbol"),
verbose = TRUE,
...
)

UpdateSymbolList(
symbols,
timeout = 10,
several.ok = FALSE,
verbose = TRUE,
...
)

Arguments
symbols A vector of gene symbols
timeout Time to wait before canceling query in seconds
several.ok Allow several current gene symbols for each provided symbol
search.types Type of query to perform:
“alias_symbol” Find alternate symbols for the genes described by symbols
“prev_symbol” Find new new symbols for the genes described by symbols
This parameter accepts multiple options and short-hand options (eg. “prev” for
“prev_symbol”)
verbose Show a progress bar depicting search progress
... Extra parameters passed to GET
202 VariableFeaturePlot

Details

For each symbol passed, we query the HGNC gene names database for current symbols that have
the provided symbol as either an alias (alias_symbol) or old (prev_symbol) symbol. All other
queries are not supported.

Value

GeneSymbolThesarus:, if several.ok, a named list where each entry is the current symbol found
for each symbol provided and the names are the provided symbols. Otherwise, a named vector with
the same information.
UpdateSymbolList: symbols with updated symbols from HGNC’s gene names database

Note

This function requires internet access

Source

https://www.genenames.org/ https://www.genenames.org/help/rest/

See Also

GET

Examples
## Not run:
GeneSybmolThesarus(symbols = c("FAM64A"))

## End(Not run)

## Not run:
UpdateSymbolList(symbols = cc.genes$s.genes)

## End(Not run)

VariableFeaturePlot View variable features

Description

View variable features

VariableFeaturePlot 203

Usage
VariableFeaturePlot(
object,
cols = c("black", "red"),
pt.size = 1,
log = NULL,
selection.method = NULL,
assay = NULL,
raster = NULL
)

Arguments
object Seurat object
cols Colors to specify non-variable/variable status
pt.size Size of the points on the plot
log Plot the x-axis in log scale
selection.method
Which method to pull. For HVFInfo and VariableFeatures, choose one from
one of the following:
• “vst”
• “sctransform” or “sct”
• “mean.var.plot”, “dispersion”, “mvp”, or “disp”
For SVFInfo and SpatiallyVariableFeatures, choose from:
• “markvariogram”
• “moransi”
assay Assay to pull variable features from
raster Convert points to raster format, default is NULL which will automatically use
raster if the number of points plotted is greater than 100,000

Value
A ggplot object

See Also
FindVariableFeatures

Examples
data("pbmc_small")
VariableFeaturePlot(object = pbmc_small)
204 VizDimLoadings

VisiumV1-class The VisiumV1 class

Description
The VisiumV1 class represents spatial information from the 10X Genomics Visium platform

Slots
image A three-dimensional array with PNG image data, see readPNG for more details
scale.factors An object of class scalefactors; see scalefactors for more information
coordinates A data frame with tissue coordinate information
spot.radius Single numeric value giving the radius of the spots

VizDimLoadings Visualize Dimensional Reduction genes

Description
Visualize top genes associated with reduction components

Usage
VizDimLoadings(
object,
dims = 1:5,
nfeatures = 30,
col = "blue",
reduction = "pca",
projected = FALSE,
balanced = FALSE,
ncol = NULL,
combine = TRUE
)

Arguments
object Seurat object
dims Number of dimensions to display
nfeatures Number of genes to display
col Color of points to use
reduction Reduction technique to visualize results for
VlnPlot 205

projected Use reduction values for full dataset (i.e. projected dimensional reduction val-
ues)
balanced Return an equal number of genes with + and - scores. If FALSE (default), returns
the top genes ranked by the scores absolute values
ncol Number of columns to display
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot objects

Value
A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

Examples
data("pbmc_small")
VizDimLoadings(object = pbmc_small)

VlnPlot Single cell violin plot

Description
Draws a violin plot of single cell data (gene expression, metrics, PC scores, etc.)

Usage
VlnPlot(
object,
features,
cols = NULL,
pt.size = NULL,
idents = NULL,
sort = FALSE,
assay = NULL,
group.by = NULL,
split.by = NULL,
adjust = 1,
y.max = NULL,
same.y.lims = FALSE,
log = FALSE,
ncol = NULL,
slot = "data",
split.plot = FALSE,
stack = FALSE,
combine = TRUE,
fill.by = "feature",
flip = FALSE
)
206 VlnPlot

Arguments
object Seurat object
features Features to plot (gene expression, metrics, PC scores, anything that can be re-
treived by FetchData)
cols Colors to use for plotting
pt.size Point size for geom_violin
idents Which classes to include in the plot (default is all)
sort Sort identity classes (on the x-axis) by the average expression of the attribute
being potted, can also pass ’increasing’ or ’decreasing’ to change sort direction
assay Name of assay to use, defaults to the active assay
group.by Group (color) cells in different ways (for example, orig.ident)
split.by A variable to split the violin plots by,
adjust Adjust parameter for geom_violin
y.max Maximum y axis value
same.y.lims Set all the y-axis limits to the same values
log plot the feature axis on log scale
ncol Number of columns if multiple plots are displayed
slot Use non-normalized counts data for plotting
split.plot plot each group of the split violin plots by multiple or single violin shapes.
stack Horizontally stack plots for each feature
combine Combine plots into a single patchworked ggplot object. If FALSE, return a list
of ggplot
fill.by Color violins/ridges based on either ’feature’ or ’ident’
flip flip plot orientation (identities on x-axis)

Value
A patchworked ggplot object if combine = TRUE; otherwise, a list of ggplot objects

See Also
FetchData

Examples
data("pbmc_small")
VlnPlot(object = pbmc_small, features = 'PC_1')
VlnPlot(object = pbmc_small, features = 'LYZ', split.by = 'groups')
Index

∗ clustering FindTransferAnchors, 81
FindClusters, 62 GetTransferPredictions, 93
FindMultiModalNeighbors, 73 IntegrateData, 100
FindNeighbors, 75 IntegrateEmbeddings, 103
FindSubCluster, 80 LocalStruct, 117
∗ convenience MappingScore, 119
DimHeatmap, 42 MapQuery, 121
DimPlot, 44 MixingMetric, 125
ReadParseBio, 151 PredictAssay, 139
ReadSTARsolo, 152 PrepSCTIntegration, 141
SpatialPlot, 189 SelectIntegrationFeatures, 184
∗ datasets TransferData, 197
cc.genes, 26 ∗ mixscape
cc.genes.updated.2019, 27 CalcPerturbSig, 23
∗ data DEenrichRPlot, 39
cc.genes, 26 MixscapeHeatmap, 126
cc.genes.updated.2019, 27 MixscapeLDA, 128
∗ differential_expression PlotPerturbScore, 137
FindAllMarkers, 59 PrepLDA, 140
FindConservedMarkers, 64 RunLDA, 159
FindMarkers, 68 RunMixscape, 161
FoldChange, 87 ∗ objects
∗ dimensional_reduction AnchorSet-class, 12
JackStraw, 107 as.CellDataSet, 14
L2CCA, 110 as.Seurat.CellDataSet, 14
L2Dim, 111 as.SingleCellExperiment, 15
PCASigGenes, 134 as.sparse.H5Group, 16
ProjectDim, 143 Cells.SCTModel, 29
ProjectUMAP, 144 CreateSCTAssayObject, 38
RunCCA, 155 DietSeurat, 41
RunICA, 157 FilterSlideSeq, 58
RunPCA, 163 GetAssay, 89
RunSPCA, 165 GetImage.SlideSeq, 90
RunTSNE, 167 GetIntegrationData, 90
RunUMAP, 169 GetTissueCoordinates.SlideSeq, 92
ScoreJackStraw, 178 IntegrationAnchorSet-class, 105
∗ integration IntegrationData-class, 106
AnnotateAnchors, 12 merge.SCTAssay, 123
FindIntegrationAnchors, 65 ModalityWeights-class, 129

207
208 INDEX

Radius.SlideSeq, 146 Radius.SlideSeq, 146

RenameCells.SCTAssay, 153 ScaleFactors, 177
ScaleFactors, 177 SlideSeq-class, 188
SCTAssay-class, 179 SpatialPlot, 189
SCTResults, 183 STARmap-class, 193
SetIntegrationData, 185 VisiumV1-class, 204
SplitObject, 192 ∗ tree
STARmap-class, 193 BuildClusterTree, 22
subset.AnchorSet, 193 ∗ utilities
TopCells, 195 AddModuleScore, 9
TopFeatures, 196 AggregateExpression, 10
TopNeighbors, 197 as.sparse.H5Group, 16
TransferAnchorSet-class, 197 AverageExpression, 18
UpdateSCTAssays, 200 CaseMatch, 26
VisiumV1-class, 204 CellCycleScoring, 28
∗ preprocessing CollapseSpeciesExpressionMatrix,
CalculateBarcodeInflections, 24 33
FindSpatiallyVariableFeatures, 78 CustomDistance, 39
FindVariableFeatures, 84 ExpMean, 51
GetResidual, 91 ExpSD, 52
HTODemux, 96 ExpVar, 52
Load10X_Spatial, 115 FastRowScale, 53
LoadSTARmap, 116 GroupCorrelation, 94
LogNormalize, 118 LoadAnnoyIndex, 116
MULTIseqDemux, 130 LogVMR, 119
NormalizeData, 132 MetaFeature, 123
Read10X, 147 MinMax, 124
Read10X_h5, 148 PercentageFeatureSet, 135
Read10X_Image, 148 RegroupIdents, 152
ReadMtx, 149 SaveAnnoyIndex, 175
ReadSlideSeq, 151 UpdateSymbolList, 201
RelativeCounts, 153 ∗ visualization
RunMarkVario, 161 AugmentPlot, 17
RunMoransI, 163 BarcodeInflectionsPlot, 19
SampleUMI, 174 BGTextColor, 20
ScaleData, 175 BlackAndWhite, 21
SCTransform, 181 CellScatter, 30
SubsetByBarcodeInflections, 194 CellSelector, 31
∗ spatial CollapseEmbeddingOutliers, 32
Cells.SCTModel, 29 ColorDimSplit, 34
FilterSlideSeq, 58 CombinePlots, 36
FindSpatiallyVariableFeatures, 78 contrast-theory, 37
GetImage.SlideSeq, 90 DimHeatmap, 42
GetTissueCoordinates.SlideSeq, 92 DimPlot, 44
ISpatialDimPlot, 106 DiscretePalette, 47
ISpatialFeaturePlot, 107 DoHeatmap, 47
LinkedPlots, 113 DotPlot, 49
PolyFeaturePlot, 138 ElbowPlot, 51
INDEX 209

FeaturePlot, 53 BlackAndWhite, 21
FeatureScatter, 56 BlueAndRed (BlackAndWhite), 21
GroupCorrelationPlot, 95 BoldTitle (SeuratTheme), 186
HoverLocator, 95 brewer.pal.info, 35, 45, 109, 191
HTOHeatmap, 97 BuildClusterTree, 22, 60, 72, 88, 126
IFeaturePlot, 100
ISpatialDimPlot, 106 CalcPerturbSig, 23
ISpatialFeaturePlot, 107 CalculateBarcodeInflections, 19, 24, 195
JackStrawPlot, 109 CaseMatch, 26
LabelClusters, 111 cc.genes, 26, 27
LabelPoints, 112 cc.genes.updated.2019, 27
LinkedPlots, 113 CellCycleScoring, 28
NNPlot, 131 CellPlot (CellScatter), 30
PlotClusterTree, 136 Cells.SCTModel, 29
PolyDimPlot, 138 Cells.SlideSeq (Cells.SCTModel), 29
PolyFeaturePlot, 138 Cells.STARmap (Cells.SCTModel), 29
RidgePlot, 154 Cells.VisiumV1 (Cells.SCTModel), 29
SeuratTheme, 186 CellsByImage, 29
SpatialPlot, 189 CellScatter, 30
VariableFeaturePlot, 202 CellSelector, 31, 46, 56
VizDimLoadings, 204 CenterTitle (SeuratTheme), 186
VlnPlot, 205 CollapseEmbeddingOutliers, 32
CollapseSpeciesExpressionMatrix, 33
AddAzimuthResults, 7 ColorDimSplit, 34
AddAzimuthScores, 8 CombinePlots, 36, 114
AddModuleScore, 9, 28 contrast-theory, 37
AggregateExpression, 10 correct_counts, 182
AnchorSet, 13, 67, 100, 101, 105, 194, 197, CreateSCTAssayObject, 38
198 CreateSeuratObject, 11, 18
AnchorSet (AnchorSet-class), 12 CustomDistance, 39
AnchorSet-class, 12 CustomPalette (BlackAndWhite), 21
AnnotateAnchors, 12
annotation_raster, 17 DarkTheme (SeuratTheme), 186
ape::plot.phylo, 136 data.frame, 16
as.CellDataSet, 14 DEenrichRPlot, 39
as.data.frame.Matrix DietSeurat, 41
(as.sparse.H5Group), 16 DimHeatmap, 42
as.Seurat.CellDataSet, 14 DimPlot, 31, 34, 36, 44, 56, 96
as.Seurat.SingleCellExperiment DimReduc, 101, 104, 198
(as.Seurat.CellDataSet), 14 DimReduc-class, 46
as.SingleCellExperiment, 15 DiscretePalette, 35, 45, 47, 109
as.sparse.H5Group, 16 DoHeatmap, 47
Assay, 102, 142, 179, 180 DotPlot, 49
Assay-class, 17
AugmentPlot, 17 ElbowPlot, 51
AverageExpression, 18 Embeddings, 195
ExpMean, 51
BarcodeInflectionsPlot, 19, 25, 195 ExpSD, 52
BGTextColor, 20 ExpVar, 52
210 INDEX

facet_grid, 112 92
facet_wrap, 112 GetTransferPredictions, 93
FastRowScale, 53 ggplot_build, 96
FeatureHeatmap (FeaturePlot), 53 Graph, 78
FeatureLocator (CellSelector), 31 Graph-class, 94
FeaturePlot, 31, 46, 53, 56, 96 Graphs, 78
FeatureScatter, 56 GroupCorrelation, 94
FetchData, 35, 45, 46, 50, 138, 206 GroupCorrelationPlot, 95
FilterSlideSeq, 58
FindAllMarkers, 59 hclust, 101, 105
FindAllMarkersNode (FindAllMarkers), 59 HoverLocator, 46, 56, 95
FindClusters, 62 HTODemux, 96, 98
FindConservedMarkers, 64 HTOHeatmap, 97, 97
FindIntegrationAnchors, 65, 101, 102, 199 HVFInfo, 99
FindMarkers, 68, 88 HVFInfo.SCTAssay, 99
FindMarkersNode (FindMarkers), 68
FindMultiModalNeighbors, 73, 129 ICAPlot (DimPlot), 44
FindNeighbors, 75 IFeaturePlot, 100
FindSpatiallyVariableFeatures, 78 image, 43
FindSubCluster, 80 IntegrateData, 12, 65, 67, 100, 105
FindTransferAnchors, 13, 81, 121, 198 IntegrateEmbeddings, 83, 103, 121, 122
FindVariableFeatures, 84, 184, 203 IntegrationAnchorSet
FindVariableGenes (IntegrationAnchorSet-class),
(FindVariableFeatures), 84 105
FoldChange, 87 IntegrationAnchorSet-class, 105
FontSize (SeuratTheme), 186 IntegrationData
(IntegrationData-class), 106
GenePlot (FeatureScatter), 56 IntegrationData-class, 106
GeneSymbolThesarus (UpdateSymbolList), Intensity (contrast-theory), 37
201 ISpatialDimPlot, 106, 191
geom_raster, 43 ISpatialFeaturePlot, 107, 191
geom_text, 112, 113
geom_text_repel, 112 JackStraw, 107, 134
GET, 201, 202 JackStrawData-class, 109
get_residuals, 91, 182 JackStrawPlot, 109, 179
GetAssay, 89
GetAssayData, 122, 198 L2CCA, 110
GetImage.SlideSeq, 90 L2Dim, 111
GetImage.STARmap (GetImage.SlideSeq), 90 LabelClusters, 111
GetImage.VisiumV1 (GetImage.SlideSeq), Labeler (LabelPoints), 112
90 LabelPoints, 112
GetIntegrationData, 90 layout, 95, 96
GetResidual, 91, 141 levels.SCTAssay (SCTAssay-class), 179
GetTissueCoordinates.SlideSeq, 92 levels<-.SCTAssay (SCTAssay-class), 179
GetTissueCoordinates.STARmap LinkedDimPlot (LinkedPlots), 113
(GetTissueCoordinates.SlideSeq), LinkedFeaturePlot (LinkedPlots), 113
92 LinkedPlot (LinkedPlots), 113
GetTissueCoordinates.VisiumV1 LinkedPlots, 113
(GetTissueCoordinates.SlideSeq), Load10X_Spatial, 115, 149
INDEX 211

LoadAnnoyIndex, 116 PurpleAndYellow (BlackAndWhite), 21

Loadings, 196
LoadSTARmap, 116 Radius.SlideSeq, 146
LocalStruct, 117 Radius.STARmap (Radius.SlideSeq), 146
LogNormalize, 118 Radius.VisiumV1 (Radius.SlideSeq), 146
LogVMR, 119 RANN, 82, 102, 199
Luminance (contrast-theory), 37 RcppAnnoy, 82
Read10X, 147
make.names, 16 Read10X_h5, 115, 148
MappingScore, 119 Read10X_Image, 115, 148
MapQuery, 83, 121 ReadMtx, 149, 151, 152
MeanVarPlot (VariableFeaturePlot), 202 ReadParseBio, 151
merge.SCTAssay, 123 readPNG, 204
merge.Seurat, 157 ReadSlideSeq, 151
MetaFeature, 123 ReadSTARsolo, 152
MinMax, 124 RegroupIdents, 152
MixingMetric, 125 RelativeCounts, 153
MixscapeHeatmap, 126 RenameCells.SCTAssay, 153
MixscapeLDA, 128 RenameCells.SlideSeq
ModalityWeights, 75 (RenameCells.SCTAssay), 153
ModalityWeights RenameCells.STARmap
(ModalityWeights-class), 129 (RenameCells.SCTAssay), 153
ModalityWeights-class, 129 RenameCells.VisiumV1
MULTIseqDemux, 130 (RenameCells.SCTAssay), 153
RestoreLegend (SeuratTheme), 186
Neighbor, 77, 78, 197 RidgePlot, 154
Neighbor-class, 131 RotatedAxis (SeuratTheme), 186
Neighbors, 78 RunCCA, 155
NNPlot, 131 RunICA, 157
NoAxes (SeuratTheme), 186 RunLDA, 159
NoGrid (SeuratTheme), 186 RunMarkVario, 79, 161
NoLegend (SeuratTheme), 186 RunMixscape, 161
NormalizeData, 132 RunMoransI, 79, 163
RunPCA, 163
patchwork, 35, 43, 45, 46, 48, 49, 56, 57, 114, RunSPCA, 165
132, 155, 205, 206 RunSVD, 82
PCAPlot (DimPlot), 44 RunTFIDF, 82
PCASigGenes, 134 RunTSNE, 167
PCHeatmap (DimHeatmap), 42 RunUMAP, 6, 145, 169
PercentageFeatureSet, 135
PlotClusterTree, 136 SampleUMI, 174
PlotPerturbScore, 137 SaveAnnoyIndex, 175
PolyDimPlot, 138, 186 ScaleData, 11, 18, 175
PolyFeaturePlot, 138, 186 ScaleFactors, 177
PredictAssay, 139 scalefactors, 204
PrepLDA, 140 scalefactors (ScaleFactors), 177
PrepSCTIntegration, 141 ScaleFactors.VisiumV1 (ScaleFactors),
ProjectDim, 134, 143 177
ProjectUMAP, 121, 122, 144 ScoreJackStraw, 110, 178
212 INDEX

SCTAssay, 99, 183 TransferAnchorSet

SCTAssay (SCTAssay-class), 179 (TransferAnchorSet-class), 197
SCTAssay-class, 179 TransferAnchorSet-class, 197
SCTModel (SCTAssay-class), 179 TransferData, 12, 81, 83, 93, 121, 122, 197
SCTransform, 141, 142, 179, 180, 181 TSNEPlot (DimPlot), 44
SCTResults, 183, 183
SCTResults<- (SCTResults), 183 umap, 172
SelectIntegrationFeatures, 66, 141, 142, UMAPPlot (DimPlot), 44
184 UpdateSCTAssays, 200
SetIntegrationData, 185 UpdateSymbolList, 9, 201
Seurat, 7, 8, 11, 19, 65, 78, 81, 102, 111, 117,
123, 142, 185 VariableFeaturePlot, 202
Seurat (Seurat-package), 6 VariableGenePlot (VariableFeaturePlot),
Seurat-class, 186 202
Seurat-package, 6 VisiumV1, 149
SeuratAxes (SeuratTheme), 186 VisiumV1 (VisiumV1-class), 204
SeuratCommand-class, 186 VisiumV1-class, 204
SeuratObject, 17, 46, 94, 109, 131, 186, 189 VizDimLoadings, 204
SeuratObject::as.Seurat, 15 VlnPlot, 205
SeuratObject::as.sparse, 16
WhiteBackground (SeuratTheme), 186
SeuratObject::Cells, 29
SeuratObject::GetImage, 90
SeuratObject::GetTissueCoordinates, 92
SeuratObject::Radius, 146
SeuratObject::RenameCells, 154
SeuratTheme, 186
SlideSeq, 151
SlideSeq (SlideSeq-class), 188
SlideSeq-class, 188
SpatialDimPlot, 58
SpatialDimPlot (SpatialPlot), 189
SpatialFeaturePlot (SpatialPlot), 189
SpatialImage-class, 189
SpatialPlot, 189
SpatialTheme (SeuratTheme), 186
split, 176
SplitDotPlotGG (DotPlot), 49
SplitObject, 192
STARmap, 117
STARmap (STARmap-class), 193
STARmap-class, 193
subset.AnchorSet, 193
SubsetByBarcodeInflections, 19, 25, 194

theme, 188
Tool, 23
TopCells, 195
TopFeatures, 196
TopNeighbors, 197