Subramanian et al.

Genome Biology (2022) 23:267 Genome Biology

https://doi.org/10.1186/s13059-022-02820-w

RESEARCH Open Access

Biology‑inspired data‑driven quality

control for scientific discovery in single‑cell
transcriptomics
Ayshwarya Subramanian1,2*† , Mikhail Alperovich1,3,4,5†, Yiming Yang1,6,7 and Bo Li1,6,7,8

†
Ayshwarya Subramanian and
Mikhail Alperovich contributed Abstract
equally to this work. Background: Quality control (QC) of cells, a critical first step in single-cell RNA
*Correspondence: sequencing data analysis, has largely relied on arbitrarily fixed data-agnostic thresh-
[email protected] olds applied to QC metrics such as gene complexity and fraction of reads mapping to
1
Klarman Cell Observatory, mitochondrial genes. The few existing data-driven approaches perform QC at the level
Broad Institute of MIT
and Harvard, Cambridge, MA,
of samples or studies without accounting for biological variation.
USA
2
Results: We first demonstrate that QC metrics vary with both tissue and cell types
Brigham and Womens’s
Hospital, Harvard Medical School,
across technologies, study conditions, and species. We then propose data-driven QC
Boston, USA (ddqc), an unsupervised adaptive QC framework to perform flexible and data-driven
3
MIT PRIMES, Massachusetts QC at the level of cell types while retaining critical biological insights and improved
Institute of Technology,
Cambridge, MA, USA
power for downstream analysis. ddqc applies an adaptive threshold based on the
4
Lexington High School, median absolute deviation on four QC metrics (gene and UMI complexity, fraction of
Lexington, MA, USA
5
reads mapping to mitochondrial and ribosomal genes). ddqc retains over a third more
Present Address: Wake
Technical Community College,
cells when compared to conventional data-agnostic QC filters. Finally, we show that
Raleigh, USA ddqc recovers biologically meaningful trends in gradation of gene complexity among
6
Center for Immunology cell types that can help answer questions of biological interest such as which cell types
and Inflammatory Diseases,
Department of Medicine,
express the least and most number of transcripts overall, and ribosomal transcripts
Massachusetts General Hospital, specifically.
Boston, MA 02114, USA
7
Present Address: Department
Conclusions: ddqc retains cell types such as metabolically active parenchymal cells
of Cellular and Tissue Genomics, and specialized cells such as neutrophils which are often lost by conventional QC.
Genentech Inc., South San Taken together, our work proposes a revised paradigm to quality filtering best prac-
Francisco, CA, USA
8
Department of Medicine,
tices—iterative QC, providing a data-driven QC framework compatible with observed
Harvard Medical School, Boston, biological diversity.
MA 02115, USA
Keywords: scRNA-seq, Quality control (QC), Data-driven, Single cell, Adaptive QC,
Exploratory data analysis (EDA), Biological variation

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Subramanian et al. Genome Biology (2022) 23:267 Page 2 of 27

Background
Single-cell RNA sequencing (scRNA-seq) offers unprecedented resolution into cell biol-
ogy by characterizing the individual cells within a biological sample of interest. Quality
control (QC) of the cells is a critical first step in any scRNA-seq data analysis, which
typically takes place after alignment of the sequencing reads to the reference genome (or
transcriptome), and generation of the cell-by-gene matrix of gene expression counts. The
goal of such “cell QC” is to remove “poor-quality” cells, based on QC metrics such as the
number of genes detected (“gene complexity” or “transcriptional diversity”), the number
of unique molecular identifiers (UMIs) recovered (typical for droplet-based technolo-
gies), and the fraction of mitochondrial and ribosomal protein genes [1]. The guiding
motivation is that tissue dissociation techniques stress the cells and as cells die, tran-
scription tapers off, cytoplasmic transcripts are degraded, and mitochondrial transcripts
dominate [2]. Thus, low complexity of genes and high mitochondrial read content have
been used as a proxy for identifying poor-quality cells (or droplets with ambient RNA).
As a corollary, high gene complexity has been used as a proxy for doublets or multiplets
in droplet-based sequencing [3]. While specialized computational strategies have been
developed for other specific QC tasks such as ambient RNA correction [4–6], empty
droplet removal [7], or doublet identification [8–10], the standard practice in “cell QC”
is to filter out cells by setting arbitrarily defined thresholds on the QC metrics. Widely
used pipelines [11, 12] by default set a flat filter on the QC criteria for each sample or
sets of samples analyzed, agnostic of the dataset and biology under study.
Although widely used, data-agnostic QC filters do not account for the fact that vari-
ation in the commonly used QC metrics may also be driven by biology (in addition to
technical factors). For example, mitochondrial transcript abundance is dependent on
cellular physiology [13], and metabolically active tissues (e.g., muscle, kidney) have
higher mitochondrial transcript content [14, 15]. Ribosomal protein gene expression has
also been shown to vary by tissue [16] in human adults and mice [17]. Although biologi-
cal variability in ribosomal protein gene expression has been reported [18], ribosomal
protein gene expression is often conflated with technical artifacts or housekeeping tran-
scription activity during analysis. Within each tissue, compartments and cell types may
show further variability in these QC attributes. For example, the total number of genes
expressed (gene complexity) varies with both cell type (cells with biologically distinct
functions) and cell state (distinct physiological functions adopted by the same cell type)
as seen during stages of mouse and human development [19]. Expression profiles also
vary with progression through the cell cycle [20] or changes in cell volume [21]. Fur-
ther, specific biological conditions or perturbations can lead to differences in these QC
measures. For example, naive poised T cells are known to have higher ribosomal content
[22, 23], as are malignant cells [24]. Activated lymphocytes such as innate lymphoid cells
(ILCs) [25] have greater transcriptional diversity, in an activation and condition-depend-
ent manner. Thus, the commonly used QC metrics can exhibit widespread biological
variability bringing to the center the biological context of the study.
The importance of calibrating cell QC for the mitochondrial read fraction based
on the mouse or human tissue of origin has been highlighted [26]; however, the pro-
posed upper limit of 5 or 10% was largely based on existing data at the time of the
study. Newer technologies (e.g., 10x v3 chemistry) may need a variable cutoff for
Subramanian et al. Genome Biology (2022) 23:267 Page 3 of 27

mitochondrial read fraction [27]. The scater package [28] encourages the use of diag-
nostic plots and sample-specific QC. More recently, probabilistic mixture modeling
has been favored for data-driven quality control at the level of samples or sample sets,
either in combination with other QC approaches [15] or standalone as in miQC [29].
However, no approach performs quality control explicitly considering the biological
variability of QC metrics at the cell type or cell-state level.
Here, we survey the variability of QC metrics across diverse scRNA-seq datasets at
the tissue and celltype level, demonstrate the need for a data-driven quality control
approach that accounts for the biological variability of QC metrics at the level of cell
types, and present a framework for data-driven QC (ddqc), inspired by unsupervised
approaches in single-cell analysis, that performs adaptive quality control while retain-
ing biological insights. ddqc partitions data by filtering out cells that fail adaptive
thresholds on QC metrics as determined by the median absolute deviation (MAD) on
each cluster of cells. Finally, we demonstrate that ddqc retains cell types that are lost
by conventional QC, expanding existing cellular taxonomies for tissues, and offering
an opportunity for further exploration and biological discovery.

Results
Survey of QC practices suggests a need for data‑driven QC
To study existing QC practices in cell filtering, we sampled 107 research papers
(“Methods”) with publication dates between 2017 and 2020, and focusing on analysis
of scRNA-seq data generated across a range of technologies (3’ 10x V2 and 3’ 10x V3,
Smartseq2, Drop-seq, mCEL-Seq2, Dronc-seq, MIRALCS, Microwell-seq) and in two
species (mouse and human [30]), and summarized the QC practices adopted (Addi-
tional file 1: Table S1). The most commonly used QC metrics were the number of
genes detected, the number of UMIs counted, and the fraction of reads mapping to
mitochondrial or ribosomal protein genes. While there were few studies that used
study-specific QC thresholds (Additional file 2: Supplementary Text), most studies
(Table 1) that applied cell QC on specific metrics used data-agnostic QC filters, usu-
ally set at 5–10% for fraction of mitochondrial reads (86% or 73 papers), and 500 for
gene complexity (86.5% or 77 papers).

Table 1 Summary of QC survey

Metric\QC Papers Data-agnostic Multiple fixed Mito or Data-driven Custom QC No
type with fixed threshold thresholds ribo genes study-level filtering
any QC (% of filtered) varying by removed before threshold
sample analysis

nCounts 65 48 (73.8%) 5 (7.7%) 0 (0%) 11 (16.9%) 1 (1.5%) 42

nGenes 89 72 (80.8%) 5 (5.6%) 0 (0%) 12 (13.5%) 0 (0%) 18
nCells 41 35 (85.4%) 3 (7.3%) 0 (0%) 2 (4.9%) 1 (2.4%) 65
%Mito 85 69 (81.2%) 5 (5.9%) 4 (4.7%) 6 (7.1%) 1 (1.2%) 22
%Ribo 7 2 (28.6%) 0 (0%) 3 (42.8%) 2 (28.6%) 0 (0%) 100
Empty droplets Doublets/multiplets Ambient RNA
4 17 6
Subramanian et al. Genome Biology (2022) 23:267 Page 4 of 27

Across species and technologies, QC metrics vary by tissue

To systematically investigate if scRNA-seq data generated by commonly used technolo-
gies retains tissue and celltype specificity of the QC metrics, we profiled QC statistics by
tissue and cell type on large public datasets after minimal basic QC (“Methods”). We sur-
veyed 5,261,652 cells from 498 samples and 47 human tissues across 34 studies [31–54],
and 966,560 cells from 337 samples and 37 mouse tissues across 5 studies [55] (“Meth-
ods”, Additional file 1: Table S2). We examined 8 human tumor types across protocols
(fresh cells/scRNA-seq vs frozen nuclei/snRNA-seq) and droplet chemistries (10x v2 vs
10x v3) [27]. A subset of the studies (Tabula Muris [56] 10X, Tabula Muris Smartseq2;
Microwell-seq mouse [57] and human [42]; Tabula Muris Senis [58]) had both uniformly
generated and processed datasets, while others (PanglaoDB [59–67]) were generated
in independent studies but uniformly processed. The mouse Tabula Muris dataset was
particularly convenient having data generated from both 3’-end droplet-based sequenc-
ing (10X, (Additional file 3: Fig. S1A, C, E)) and full-length RNA plate-based Smartseq2
techniques (Additional file 3: Fig. S1B, D, F) from the same samples, and processed uni-
formly using the same reference and computational pipelines.
We found a tissue-specific (Fig. 1) trend for the QC metrics across studies. In general,
we found variation by tissue for proportion of mitochondrial reads (Fig. 1A, B) within
the same study regardless of the technology used (Tabula Muris 10X, Tabula Muris
Smartseq2; Microwell-seq mouse and human) with some tissues emerging as having
higher mitochondrial content (e.g., kidney, colon, heart, liver). The tissue-specific order-
ing of mitochondrial reads seen in [13] was most faithfully recapitulated by the Smart-
seq2 dataset (Additional file 3: Fig. S1B) with kidney, colon, cerebellum, and heart having
the highest mitochondrial load. Differences in the gene complexity (Fig. 1C, D) and the
percent of ribosomal protein genes (Fig. 1E, F) were also observed among tissues. Across
both Tabula Muris 10X and Tabula Muris Smartseq2, the tongue had the highest mean
gene complexity (Additional file 3: Fig. S1C, D), with the mean percentage of riboso-
mal protein reads being higher in the 10X dataset (Fig 1E). Trends were generally also
maintained with age (Tabula Muris Senis 30m, Additional file 3: Fig. S2A, C, E). When
compared to frozen tumor nuclei, the gene complexity was higher for cells (Additional
file 3: Fig. S2D). Further, within each tissue, multiple density modes were evident (Fig. 1)
for the QC metric studied. Finally, we note that the summary statistics of the QC metrics
can vary by the experimental condition (technology and study) on the same tissue.

Across species and technologies, QC metrics vary by cell type within a tissue
We next assessed cell subset-specific (representing cell types or cell states) QC attrib-
ute differences within tissues by uniformly processing all datasets (starting with the
gene expression count matrices) to derive clusters within each tissue without applying
standard QC cutoffs (“Methods”). However, many publicly available datasets did not
come with assigned celltype annotations. To uniformly assign biological annotations
to the cell clusters, we devised a heuristic score function leveraging the top differen-
tially expressed genes in a cluster, and the PanglaoDB [59] database of marker genes
to predict the most probable cell-type annotation. We tested the annotation strategy
on 4 mouse (Tabula Muris Smartseq2, Tabula Muris 10X, Tabula Muris Senis 24
Subramanian et al. Genome Biology (2022) 23:267 Page 5 of 27

Fig. 1 QC metrics vary by tissue. (X-axis) Fraction of mitochondrial reads (A, B), gene complexity (C, D), and
percentage of ribosomal protein genes (E, F) per cell across human tissues (Y-axis) and technologies. Various
human tissue scRNA-seq datasets generated by 10X droplet-based (A, C, E) and Microwell-seq (B, D, F)
technologies. Each row in a panel is a density curve with the mean represented by a blue diamond. Red lines
indicate conventional threshold values set at 10% for percentage of mitochondrial reads, and 200 for gene
complexity

months, Tabula Muris Senis 30 months) and 1 human (Human Tissue Atlas) data-
sets which had partial annotations provided by the authors. On these data, our heu-
ristic approach had an accuracy of 80.2 and 92.1% for cluster annotations in human
and mouse data respectively (Additional file 1: Table S3, “Methods”). We applied our
Subramanian et al. Genome Biology (2022) 23:267 Page 6 of 27

heuristic approach to all test datasets and then examined trends of the QC metrics
among cell types within tissues. As case studies, we manually verified annotations and
describe examples for murine (Additional file 3: Fig. S3) and human tissues (Fig. 2).
Across all tissues, we observed variability by annotated cell type, in the per cell QC
metrics (fraction of mitochondrial and ribosomal reads mapped, and gene complexity
per cell). To illustrate the impact of standard practice QC thresholds, we applied QC
thresholds of 10% for the maximum mitochondrial read fraction and 500 genes detected

Fig. 2 QC metrics vary by celltype. (X-axis) Fraction of mitochondrial reads (A, B), gene complexity (C, D),
and percentage of ribosomal protein genes (E, F) per cell across cell types (Y-axis) of various human tissues:
kidney (A), testis (B), adipose (C), substantia nigra (D), bone marrow (E), and lung (F). All scRNA-seq data was
generated using the 10X droplet-based technology. Each row in a panel is a density curve with the mean
represented by a blue diamond. Red lines indicate conventional threshold values set at 10% for percentage
of mitochondrial reads, and 200 for gene complexity. Cluster numbers are indicated preceding the cell type
annotation
Subramanian et al. Genome Biology (2022) 23:267 Page 7 of 27

for minimum gene complexity. A fixed cutoff of 10% mitochondrial read fraction led to
loss of parenchymal cell subsets in human kidney and testis (Fig. 2A, B), and mouse cer-
ebellum, and colon (Additional file 3: Fig. S3A,B). More broadly, mitochondrial-read-
rich clusters ranged from muscle cells to tissue-parenchymal cells such as enterocytes
(gut), proximal tubular cells (kidney), or sertoli cells (testis), all cell types known to have
high metabolic activity and energy needs such as active transport in the kidney proximal
tubule, and oxidative phosphorylation in cardiomyocytes of the heart. Even a conserva-
tive fixed cutoff of 200 genes led to loss of diverse cell subsets including immune cells
such as neutrophils (Additional file 3: Fig. S3C, D) and neurons (Fig. 2D). Cell type-spe-
cific trends in percent ribosomal protein genes were also evident (Fig. 2E, F, Additional
file 3: Fig. S3E,F). Thus, data-agnostic thresholds remove biologically relevant cells, and
hence, QC based on these metrics must not only adapt to different tissues or samples
but also to cell states and cell types.

ddqc: a cell‑state adaptive quality control framework

To account for biological variability among QC metrics, and also adapt to differences
in experimental conditions (study design, technology, etc.), we propose data-driven QC
(ddqc, Fig. 3A), an unsupervised, data-driven, and adaptive thresholding framework
for optimal capture of biological diversity. Inspired by and adapting existing unsuper-
vised approaches in scRNA-seq analysis [68], ddqc identifies neighborhoods of cells by
graph-based clustering and performs QC on these clusters using an adaptive threshold-
ing approach. The basic concept is that data must be partitioned by biology and that QC
must be performed on these independent partitions. Briefly, cells that pass empty drop-
let filters are subjected to dimensionality reduction by principal component analysis,
followed by nearest neighbor graph construction and clustering to identify cell clusters
with similar transcriptional states (details in “Methods”). Our approach does not rely on
prior annotation, rather it identifies biologically similar cells based on the density of the
transcriptional data. Within each such cluster, we identify “outliers” based on one- or
two-sided thresholds on the QC metric of interest, defined as those cells that lie beyond
a chosen number of median absolute deviations (MAD) from the cluster QC metric dis-
tribution median. Cells that pass these thresholds then enter downstream analysis.
The specific downstream analysis depends on the study and biological questions of
interest. For example, the next step may range from integration with other data modali-
ties (e.g., spatial data) or batch effect correction or cell classification. If the next step is
indeed conventional analysis involving clustering-based cell-type/state identification,
followed by differential gene expression, analysts may choose to start with the cluster-
ing labels that ddqc generates during QC (and returns as an output) to merge, re-cluster,
or subcluster based on their research question. ddqc is available as both R and Python
packages on GitHub and can be readily plugged into standard scRNA-seq analysis pipe-
lines such as Pegasus [69] or Seurat [12]. Flexible options and exploratory plots are
provided to the user for more control. ddqc is agnostic to the approach used to remove
empty droplets (Additional file 2: Supplementary Text) which can be user defined. Our
results were robust to varying clustering algorithms (Additional file 3: Fig. S4A, B, Addi-
tional file 1: Table S4) or hyperparameters at the different steps (Fig S4C,D). Cluster-
ing approaches perform on par with automated cell-type annotation methods [70–72]
Subramanian et al. Genome Biology (2022) 23:267 Page 8 of 27

Fig. 3 ddqc retains biologically meaningful cells that conventional QC filters out. A Overview of the ddqc
approach. B, C ddqc retains more cells when compared to the standard cutoff approach across B tissues in
the Tabula Muris dataset, and C scRNA-seq data generating technologies. D UMAP visualization of Tabula
Muris lung cells. Colors represent whether the cells are included in the paper or uniquely retained by ddqc.
E Violin plot visualization of cell type-specific signature scores in average log(TPX+1). From top to bottom:
muscle, neutrophil, NK cells, and Gamma-delta T cells. F UMAP visualization of joint clustering of cells retained
by both ddqc and the standard cutoff in the Tabula Muris heart and aorta tissues. G Proportion of cells
retained by ddqc, standard cutoff, or both in the mouse heart and aorta tissues

(Additional file 1: Table S5). To help evaluate the MAD multiplier parameter to use as
the adaptive threshold, ddqc provides exploratory plots. Our extensive evaluation sup-
ports an analyst involved interactive analysis that integrates EDA and the analyst’s
expertise in the problem of interest.
We evaluated the performance of ddqc on all test datasets (Additional file 1: Table S2)
applying adaptive QC on three QC metrics: fraction of UMIs mapped to mitochondrial
Subramanian et al. Genome Biology (2022) 23:267 Page 9 of 27

genes, gene complexity, and number of UMIs. For comparisons, we ran conventional
QC (“standard cutoff ”) on our test datasets using a fixed threshold of 10% as the maxi-
mum fraction of mitochondrial reads, and 200 as the minimum gene complexity. We
then evaluated the cells that passed QC by either approach in a number of ways: ability
to (1) improve power, (2) expand existing cellular taxonomies, (3) recover biologically
meaningful states, and (4) discover broadly useful insights of transcriptional activity.
At instances, we use the terms cell “states” and “types” interchangeably as there may
be multiple clusters with identical celltype markers, potentially representing biological
states.

ddqc improves power for downstream analysis when compared with conventional QC
methods
We computed the number of cells retained by either ddqc or conventional QC and deter-
mined the breakdown by QC attributes. ddqc preserved more cells in comparison to
conventional QC across datasets and biological conditions (Additional file 1: Table S6).
Overall, ddqc retained up to a median of 95.4% of input cells versus 69.4% cells using the
standard cutoff approach. The higher number of cells retained by ddqc held across tis-
sues (Fig. 3B) and technologies (Fig. 3C). Stratified by QC attributes, on average 83.19%
of cells lost by ddqc are due to thresholds on the proportion of mitochondrial reads
while 6.2% are lost due to gene complexity (Additional file 1: Table S6) thresholds. Thus,
the higher number of cells preserved by ddqc provides more statistical power for down-
stream analysis.

ddqc retains biological cellstate information lost using default cutoff or data‑driven
approaches that do not consider biology
As ddqc applies QC per cluster, it helps retain several cell types or states of biological rel-
evance. We illustrate the biological relevance of ddqc in two ways. First, using the Tabula
Muris lung dataset as a case study, we compared changes in lung cell taxonomies derived
by conventional clustering analysis following either ddqc or the author-defined cutoffs.
In the Tabula Muris paper, the authors used fixed cutoffs of 500 genes for minimum
gene complexity and 1000 UMIs for the minimum number of UMIs. After QC by ddqc,
we overlaid cell barcode annotations (Fig. 3D) provided by the authors [56] to define
clusters with cells retained both in the paper and ddqc, and those exclusively retained
by ddqc (i.e., all cells in the cluster were filtered out in the paper but retained by ddqc).
Examining clusters exclusively retained by ddqc, we find various cell types of interest
such as muscle cells, neutrophils, Natural Killer (NK) cells, and T cells, which we vali-
date using their known canonical signatures (Fig. 3E, Additional file 1: Table S7). These
cell states were filtered out in the Tabula Muris study and not analyzed downstream.
When these data are lost, we also lose the biology or insights we might have learned by
analyzing them. Thus, using ddqc, we are able to expand tissue cellular taxonomies by
retaining tissue-native cell types missed by arbitrary cutoff-based QC.
Next, to demonstrate that ddqc recovers biologically meaningful states, we proceeded
to annotate the cells that passed QC using our heuristic annotation strategy. Since our
annotation strategy labels cell clusters and not individual cells, we jointly clustered the
cells retained by both ddqc and the standard cutoff QC, and then applied our heuristic
Subramanian et al. Genome Biology (2022) 23:267 Page 10 of 27

clustering strategy to assign biologically relevant labels. To evaluate differences in the

filtered cells by both approaches, we defined “uniquely retained” clusters as those that
had at least 30 cell members, and 85% of cluster membership consisted of cells uniquely
retained by either QC method. No cluster was unique to the standard cutoff approach
by the above definitions whereas several biologically meaningful clusters were uniquely
retained by ddqc (Additional file 1: Table S8). We describe three examples: Tabula Muris
heart and aorta (Fig. 3F, G, Additional file 3: Fig. S4E, G), human Olfactory Epithelial
cells (Additional file 3: Fig. S4F,H, S5A,B), the human lung (Fig S5C,D). Compared to
the standard cutoff method, ddqc retained cell subsets with low gene complexity includ-
ing olfactory epithelial cells, dendritic cells, erythroid precursor cells, and platelets
which were filtered out by the conventional QC approach. Cardiomyocytes (Additional
file 3: Fig. S3A) and lung muscle (Fig. 3G) cells were mito-rich and retained in ddqc.
The majority of cells with high mitochondrial content are diverse epithelial cells in both
mouse and human. We provide a table of cell states lost when conventional methods are
used across all our surveyed datasets (Table S8).
Finally, to compare with a data-driven approach, we ran miQC using standard set-
tings (“Methods”) on the human olfactory epithelium and the mouse heart datasets
(Additional file 3: Fig. S5E, F). For the human Olfactory Epithelium, both ddqc and
miQC retain all clusters (miQC retaining up to 95% of cells as ddqc) with ddqc retaining
more of mito-rich olfactory epithelial cells. However, in the Tabula Muris mouse heart
example (Additional file 3: Fig. S5E), miQC retained only 90.5% of cells as ddqc, com-
pletely removing the cardiomyocyte cluster. The cardiomyocyte cluster had a median of
15.178% reads mapping to mitochondrial genes, and 2427.67 as the median gene com-
plexity, which ddqc retains. Cardiomyocytes are essential parenchymal cells of the heart.
In both examples, miQC retained fewer cells exclusively (that ddqc did not); however,
these did not map to a missing biologically relevant cell type. Thus, ddqc retains biologi-
cally relevant cell types that miQC filters out.

Which cells have the least and most number of transcripts?

We next turned to insights such as patterns of celltype-specific gene usage that a more
biology-driven QC approach such as ddqc preserves (Fig. 4). Following application of
ddqc, we examined trends in QC metrics (Additional file 1: Table S9), to answer ques-
tions such as “which cell types or states transcribe the fewest or largest number of
genes?”. We defined cell states with low gene complexity as those with both low median
number of genes detected (< 200) as well as low median percentage of mitochondrial
reads (<10%). Across 20 human studies and 159 clusters, 44 of the clusters (27.7%) sat-
isfying the criteria were diverse immune cells including dendritic cells, plasma cells, T
cells, NK, and mast cells. Other subsets included endothelial subsets, platelets, and RBCs
(6%). Specific parenchymal cells with low gene complexity were specialized cells such as
gastric chief cells (PGA5+, PGC+, CHIA+, PGA3+, LIPF+) of the stomach, cardiomyo-
cytes (NPPA+, NACA​+, NACA2+, MYL2+), neuronal subsets (Schwann, astrocytes, neu-
rons) of the substantia nigra, and olfactory epithelial cells. Across 4 large mouse studies
and 465 clusters, 133 (28.6%) were immune cell clusters including 28 neutrophil (Elane+,
Prtn3+, Mpo+) subsets, 27 B cells, and 46 macrophage/Kupffer subsets. Endothelial (46)
Subramanian et al. Genome Biology (2022) 23:267 Page 11 of 27

Fig. 4 Schematic summarizing trends in “low” and “high” transcriptional diversity among mouse and human
cell types for total number of genes (A) and ribosomal gene fraction (B). Top row represents the most
prevalent cell type within the group (% of total clusters examined) for mouse and bottom row for human.
Cell types are further partitioned within the immune cell category. A Gene complexity trends. Cell types
with number of genes < 200 median number of genes detected are in the low gene complexity group while
those with > 2000 median number of genes detected are in the high category. B Ribosomal gene fraction
trends. Cell types with median fraction of ribosomal genes < 10% are in the low gene complexity group while
those with median fraction of ribosomal genes > 20% are in the high category
Subramanian et al. Genome Biology (2022) 23:267 Page 12 of 27

and erythroid (23) lineages followed. Parenchymal cells included lactating and involut-
ing mammary gland cells, pancreatic acinar cells, and diverse epithelial cells.
Next, we looked at cell types/states with high gene complexity (> 2000 median genes,
< 10% fraction mitochondrial reads). Among 311 such clusters in humans, neurons (35),
and fibroblast (28) emerged as the higher ranked ones, along with epithelial cells (159).
In mice, across 377 clusters, macrophages/microglia (61), fibroblasts (53), neuronal (20),
and diverse epithelial cells (111) were among the most populous subsets with high gene
complexity.

Immune cells have a high fraction of ribosomal protein content

Examining trends of ribosomal protein transcription, we defined high or low median
ribosomal protein gene complexity as that with greater than 20% reads or lower than
10% reads mapping to ribosomal protein genes, and lower than 10% reads mapping to
mitochondrial genes. Among 438 human clusters with high ribosomal protein gene
complexity, 212 (48.4%) were immune cell subsets including 85 T cell, and 50 dendritic
cell subsets. Immune cell function often requires rapid protein translation [23, 73].
Other preponderant subsets were epithelial (110) and fibroblasts (43). Among 450 such
clusters in mice, 241(53.6%) were annotated as immune including diverse subsets (B cell
(78), macrophages (44), and T cells (75)) suggesting that certain immune states may have
high translational activity and need for ribosomal protein genes.
Neurons (20.6%) were a large fraction of human cell states with lower ribosomal pro-
tein gene complexity. In mouse, cell states with low ribosomal protein gene complexity
included diverse epithelial and immune cells, fibroblasts, and endothelial cells. Thus, a
more context-focused QC approach such as ddqc can enable us to recapitulate and study
fundamental patterns in cell-type-specific gene expression and associated function.

Discussion
Cell quality control remains an essential step in scRNA-seq data analysis; however, con-
ventional approaches apply arbitrary filters on defined QC metrics without accounting
for the biological context. The standard QC practice among published papers is largely
data-agnostic and arbitrary threshold-based. We have demonstrated (Figs. 1 and 2) that
not accounting for the underlying biological heterogeneity at the level of cell states dur-
ing QC can lead to loss of relevant biological insights (including important cell types)
as well as reduced statistical power for downstream analysis. However, identifying cell
types and cell states is a time-consuming process requiring either well-annotated train-
ing sets or involves the manual and subjective task of cell-state annotation. The field of
single-cell biology is still in the nascent stages of building experimentally validated and
reproducible ontologies of cell states. The few existing automated annotation strategies
are limited in the number of tissues they can handle. To overcome these challenges, we
present an unsupervised approach ddqc that leverages clustering to identify transcrip-
tionally similar cellular neighborhoods (approximating broad cell types) and performs
adaptive QC on these clusters. The unsupervised approach underlying ddqc performs
on par with independent annotation strategies on test datasets (Additional file 2: Sup-
plementary Text).
Subramanian et al. Genome Biology (2022) 23:267 Page 13 of 27

We observe limitations of our approach: (1) ddqc applies adaptive thresholds on each
cluster, and hence, we are likely to lose a small number of good-quality cells (false posi-
tives) due to inherent spread of the cluster data distribution. (2) While in most cases,
ddqc retains clusters that are biologically meaningful, in some cases, ddqc may retain
cells (Additional file 1: Table S8) with high percentages of mitochondrial genes that may
be a mix of biology and technical artifacts. These clusters when sub-clustered do not
always represent bimodal distributions (Additional file 3: Fig. S3), rather a gradation, and
there is no perfect way to assess the right cutoff. Such cells are usually subsets of larger
neighborhoods of biologically meaningful cells that reflect true metabolic stress due to
the biological condition studied. In the current version of ddqc, removal of such cells
has been left to the analyst after examination via Exploratory Data Analysis (EDA) in
the context of the biology of the study, and during downstream analysis. We believe QC
should be iterative and to help empower the user, ddqc provides detailed statistics for all
cells that pass or fail adaptive QC.
ddqc provides several advantages relative to conventional cutoff or biology-agnostic
data-driven approaches. First, it retains more cells than standard or data-driven QC
approaches leading to more power for downstream analysis. Second, the additional cells
retained by ddqc are biologically meaningful thus increasing the potential for further
biological discovery. Such biological insights include retaining a diversity of cell types
with extreme value QCs and rare cells, as well as uncovering study-specific metabolic
and physiological programs that may dictate changes in these common QC metrics.
Further investigation of retained cell states may provide insights into the underlying
biological processes. Finally, we examine cells lost by conventional QC to add insights
into questions of fundamental interest in biology such as parsimony in total gene usage
or transcription. Our analysis has revealed interesting biological observations in terms
of overall transcriptional diversity of cell states, as well as ribosomal protein gene
expression.

Conclusions
In summation, we propose a biology-centered and iterative approach to cell quality
control for scRNA-seq data that retains cell states of critical biological relevance often
removed by conventional QC. By contributing a framework for quality control that con-
siders the biological properties of data, ddqc can revise how data analysis is performed
in every scRNA-seq study.

Methods
QC survey
We conducted a survey of 107 single-cell and single-nucleus RNA sequencing papers
published between 2017 and 2020. Papers included in the survey were collated either
from Twitter posts, searches on Google, or a curated scRNA-seq database [74]. For each
paper, we recorded the Quality Control (QC) strategy from the “Methods” section into
Additional file 1: Table S1. Additional information was also recorded for each paper,
including:
Subramanian et al. Genome Biology (2022) 23:267 Page 14 of 27

• Year published
• Organism
• Tissue of origin
• Sequencing technology
• Analysis software
• Preprocessing software

QC was classified into the following categories:

• QC to remove low-quality cells and genes by QC metric

° Number of counts
° Number of genes
° Percent of mitochondrial transcripts
° Percent of ribosomal transcripts
° Number of cells in which gene is present

• QC to remove empty droplets

• QC to remove doublets/multiplets
• QC to account for ambient/background RNA

We categorized the papers based on the type of QC used for a particular metric.
These categories were:

• Data-agnostic fixed threshold—QC removed all cells with a metric above/below

a certain number (for example keep all cells with <10% mitochondrial tran-
scripts)
• Multiple fixed thresholds—several fixed thresholds for different samples
• Data-driven study-level threshold—QC threshold was determined from the
data (for example, keep all cells with a number of genes within 2 SDs from the
median)
• Custom—QC that was very specific for the particular paper
• No filtering—no filtering based on this metric was done

The summary of the QC survey and QC methods are documented in Table 1 and
the “Results” section.

Datasets
We downloaded publicly available mouse (n=5) and 32 human (n=32) (Table S2)
single-cell (scRNA-seq) or single-nucleus (snRNA-seq) RNA sequencing datasets.
We restricted our study to droplet- (10X Genomics), Microwell-seq, and plate-based
(SmartSeq2) technologies from various tissues.
We downloaded data at the level of gene counts after preprocessing (genomic refer-
ence alignment and gene-level quantification) but prior to any quality control (QC).
However, many datasets in public repositories were already filtered using cutoffs or
Subramanian et al. Genome Biology (2022) 23:267 Page 15 of 27

were aligned to reference genomes with missing genes. In some cases, we were able
to contact study authors (e.g., Tabula Muris) and get the unfiltered expression matri-
ces. Links to the unfiltered datasets used can be found in Additional file 1: Table S2.
Our dataset search was agnostic to the computational preprocessing methodology or
genome reference version used.

Input files
For all analyses, we start with loading the unfiltered or raw cell-by-gene matrix stored
either in the mtx, csv, txt, or h5ad format.

ddqc
We propose an adaptive thresholding method to perform quality control at the level
of cell types, thus taking into account differences between them. The first step of this
method is to cluster the cells using standard scRNA-seq analysis preprocessing and
clustering steps. We assume that within each cluster, cells are of the same or closely
related cell type with shared biological properties. In each cluster, we expect out-
liers—cells with the number of UMI counts, number of genes, or percent of mito-
chondrial transcripts significantly different from the cluster average. We assume that
those differ in quality from other cells in their cluster and remove them by calculat-
ing a cutoff for each cluster based on median absolute deviation and a user-defined
parameter x. We chose the median absolute deviation (MAD) to be a more robust
statistic to define outlier thresholds instead of the zscore which assumes normality,
or IQR which is less permissive. If the cell has a value higher (percent.mito) or lower
(n_counts, n_genes) than x MADs from the median in its cluster, this cell will be fil-
tered out; all remaining cells will be sent for downstream analysis. If the cluster ddqc
threshold was bigger than 200 n_genes, or lower than 10% mito, we would set it to
200 or 10 respectively.
ddqc uses preprocessing and clustering functions provided by the Pegasus (https://​
pegas​us.​readt​hedocs.​io/) for the Python package: https://​github.​com/​ayshw​aryas/​
ddqc. An R package using functions in Seurat is also available: https://​github.​com/​
ayshw​aryas/​ddqc_R.
Our pipeline starts with a loading of the unfiltered cell-by-gene matrix stored either
in mtx, csv, txt, or h5ad format. Below, we list the Python Pegasus functions with the
Seurat R functions in parenthesis.

• Initial or Empty droplet Filtering: by default, a minimal filtering is conducted

to remove obvious low-quality cells or empty droplets: cells with less than 100
genes or with more than 80% of mitochondrial transcripts were removed using the
Pegasus functions qc_metrics and filter_data (subset in R). Users may choose to
skip the step, provide their own filters for each QC metric or provide filtered input
files after applying an empty droplet detection method of their choice. For all anal-
ysis and results, initial filtering was conducted to remove poor-quality cells: cells
with less than 100 genes or with more than 80% of mitochondrial transcripts and
genes present in less than 3 cells are removed. The Initial Filtering step is essential
Subramanian et al. Genome Biology (2022) 23:267 Page 16 of 27

for computational efficiency as otherwise, we may have on the order of a million

or more barcodes in case of droplet-based scRNA-seq.
• Normalization is performed using the function NormalizeData (NormalizeData
in Seurat): normalize the feature expression measurements for each cell by the
total expression, multiply by a scale factor (10,000), and log-transform the result
to get log(TPX+1) values.
• We find the top 2000 highly variable genes using the function call highly_varia-
ble_features (FindVariableFeatures in Seurat). We scale the expression matrix of
highly variable genes: shift the expression of each gene so that the mean expres-
sion across cells is 0 and scale the expression of each gene so that the variance
across cells is 1 (In Pegasus, scaling is part of pca, in Seurat ScaleData)
• Next, dimensionality reduction is performed using principal component analysis
(PCA) using pca (RunPCA) with the number of principal components set at 50.
• Graph-based clustering of cells was performed by first building the k-nearest
neighbor graph setting k=20 [75], and then the Louvain algorithm for cluster-
ing [76] or community detection with the resolution set at 1.4 using the functions
neighbors (FindNeighbors) and louvain (FindClusters) functions.
• Then we iterate through each of QC metrics to determine the cutoff values:

° First we create a true/false numpy array (vector in R) that would represent

whether the cells have passed ddqc
° For each cluster, we find lower (for n_counts and n_genes, otherwise set to
negative infinity) and upper (percent mito, otherwise set to positive infinity)
cutoff (median ± x × MAD). x is user defined with a default of 2. For number
of genes: If lower cutoff is more than 200 genes, it would be set to 200 (by
default)

– For percent mito: if upper cutoff is less than 10R%, it would be set to 10
(by default)

° Finally, if the cell is outside the bounds defined by cutoffs, it would be

marked as false in the ddqc array

• We do an AND operation between all ddqc metric-specific arrays. Cells that are
marked as true in this array have passed ddqc and are retained for downstream
analysis

In the Pegasus and Seurat workflows, in addition to returning the filtered object,
ddqc returns a pandas dataframe with the following information for each cell:

• True/false value that indicates whether the cell passed the ddqc
• Cluster number that was assigned to this cell in the initial clustering
• For each QC metric:

° The metric itself

° Lower cutoff (cluster median − 2 cluster MAD) for this metric for the cell’s
cluster. If there is no cutoff, this field will be equal to None
Subramanian et al. Genome Biology (2022) 23:267 Page 17 of 27

° Upper cutoff (cluster median + 2 cluster MAD) for this metric for the cell’s
cluster. If there is no cutoff, this field will be equal to None
° True/false value that indicates whether the cell passed the ddqc for the
given metric

In addition, the ddqc workflow displays four plots for exploratory data analysis:

• Two boxplots: one shows the percent mito by cluster with a red line at 10% that indi-
cates the standard fixed threshold for percent mito, and the other shows the log2 of
the n_genes by cluster with a red line at 200 genes (7.64 in log2-scale) that indicates
the most commonly used fixed threshold for number of genes.
• If the MAD was selected as the threshold calculation method and the MAD multi-
plier was set using the threshold parameter of the ddqc_metrics function only, ddqc
will generate two facet plots that show how the number of cells that are filtered out
changes depending on the threshold value. These plots will help you to pick a thresh-
old parameter if you want to tune it.

Automated cell‑type annotation

We automated the task of mapping cell type annotations to clusters using the PanglaoDB
cell-type gene expression signatures as the reference dataset. Using the PanglaoDB cell-
type:marker mappings, cell-type labels were assigned for each cluster as follows:

(1) We computed cluster-specific differentially expressed genes (DGE) by testing for

genes differentially expressed in the cluster of interest vs all else. For the testing,
we used the default differential expression test used in Seurat for the R version or
Pegasus for the Python version.
(2) We filtered the DEG to retain those genes with at least a log fold change of > 0.25,
percent expressed in the cluster of interest > 25%, and q_value < 0.05.
(3) We iterated through each cluster to assign cell-type scores as follows:

a. First, we iterated through the filtered DEG of the current cluster to check for
matches in PanglaoDB.

i. If there was an entry that the gene indicates for a particular cell type, the
average log fold change of that gene was added to the score of the cell type.
ii. Only cell-type annotations which included at least three such marker
genes were retained

b. The cluster was assigned the cell-type annotation with the highest score. Other-
wise, the cell type would be stated as Unknown.

We note that the accuracy of our method is contingent on the accuracy of markers
in the PanglaoDB dataset which would get updated on a regular basis. The PanglaoDB
markers database does not have enough genes for certain cell types, which causes them
to be assigned to similar but not identical cell types (For example, macrophages which
Subramanian et al. Genome Biology (2022) 23:267 Page 18 of 27

are antigen-presenting cells (APC) are often labeled as dendritic cells, another APC). For
examples in Figs. 2 and 3, annotations were manually verified.

Automated cell‑type annotation accuracy assessment

In order to assess the accuracy of our cell-type annotations method, we have com-
pared the results of automated annotations with the annotation provided by the pub-
lisher of the dataset, if such annotation was provided. Datasets where the authors
provided annotations included the Human Tissue Atlas; human adipose (inhouse
annotated), heart, and lung; Tabula muris (10x), Tabula muris (Smartseq-2), Tabula
senis 10x 24 and 30 month. The accuracy was calculated using the steps below.

(1) First, we annotate the clusters after just the default empty droplet filters. We do it
by mapping the annotation that is the most frequent among the cells of the clus-
ter. If most of the cells do not have an annotation, the cluster will be marked as
“unknown”.
(2) For accuracy analysis, we are only including the clusters that had an annotation
(not “unknown”) and where at least 75% of cluster cells had that annotation.
(3) For the comparison, we have established a number of pairs of annotations that we
considered to be the same (Additional file 1: Table S3). Some of these pairs are just
different in naming between predicted and author-provided annotations (exam-
ple NK cells VS Natural Killer cells), and others were validated by marker genes
to be more accurately defined using our strategy than the author-defined annota-
tions (e.g., cluster 6 in our analysis of the Tabula Muris Smartseq2 kidney dataset
were author annotated to be collecting duct cells when they highly expressed loop
of Henle and distal tubular markers Umod and Slc12a1, and which was correctly
predicted by our algorithm).
(4) Then, we count the number of clusters with a mismatch between automated
annotation and the annotation provided by the publisher. If the annotation pair is
included in the table from step 3, it will not be counted as a mismatch. After that,
we compute the accuracy percentage.

The tables of the same cell types, mismatches, exact numbers, and breakdown by
the dataset are provided in Additional file 1: Table S3.

Comparison of ddqc with author‑provided annotations

We have compared ddqc with author-provided quality control in Tabula muris tissue
(Fig. 3):

(1) First, the author-provided annotations were downloaded from figshare (https://​
figsh​are.​com/​artic​les/​datas​et/​Single-​cell_​RNA-​seq_​data_​from_​micro​fluid​ic_​emuls​
ion_​v2_/​59689​60?​file=​13088​039).
(2) Then we calculated the percent of cells exclusive to ddqc in each cluster after ddqc
filtering (Additional file 1: Table S6). It was calculated by taking the number of cells
whose barcodes were not present in author annotations (which means they were
Subramanian et al. Genome Biology (2022) 23:267 Page 19 of 27

not included by the author for final analysis) and dividing it by the total number of
cells in the cluster.
(3) To verify the automated annotation for clusters where the number of cells exclu-
sive to ddqc was 100%, we have computed signature scores for each of the clus-
ters (using the “pegasus.calc_signature_score” function) with cell-type markers
(Fig. 3E). You can find the signature genes in the Additional file 1: Table S6.
(4) We have also generated UMAP plots with cells colored based on percent exclu-
sive of their cluster. We had 2 categories: fully exclusive to ddqc or shared with the
paper (Fig. 3D)

Comparison of ddqc to the standard cutoff method

We compared ddqc with the standard cutoff or static threshold method (default in most
pipelines) as a control, and only empty droplet filtering for reference:

(1) ddqc using the same steps as described in the ddqc section for loading the data and
filtering.
(2) Standard cutoff or static threshold (cells with number of genes less than 200 and
mitochondrial transcripts percent higher than 10% are removed regardless of filter-
ing)
(3) No QC (done for reference)

First, we evaluated the retained cells in all the three approaches independently by
graph-based clustering, followed by differential gene expression using de_analysis func-
tion and UMAP visualization using umap the function. Also, additional statistics were
recorded for future analysis (Information about clusters and cells). Exploratory data
analysis (EDA) was performed by generating summary plots including boxplots, joy-
plots, and colored UMAP plots.
Next, for comparisons, we performed joint clustering as follows:

(1) After QC was performed, each barcode is assigned a label which indicates if it was
filtered or retained by each method. Possible options are retained by both methods,
retained by ddqc only, retained by cutoff only, neither (removed by both cutoff and
ddqc)
(2) Barcodes that were marked as “neither” were removed
(3) All remaining barcodes were clustered (as above) and visualized using UMAP.
(4) Both cluster and filter labels were used to color the UMAPs for exploratory data
analysis. Barplots were also generated per cluster to visualize the distribution of
each cluster by cell retained in each method.
(5) DGE was performed on the clusters to assign cell identity and to identify cell types
lost by single-threshold QC.

These plots helped to demonstrate differences between static threshold and ddqc by
highlighting clusters of cells that were kept by one method but lost by another.
Subramanian et al. Genome Biology (2022) 23:267 Page 20 of 27

Unique clusters
To demonstrate differences between static threshold (“cutoff ”) and ddqc, we determined
how many meaningful “unique” clusters ddqc retained. A “unique” cluster was defined as
a cluster with at least 30 cells, and with at least 85% of its cells retained only by ddqc but
filtered out by cutoff method. The presence of unique clusters indicates that a popula-
tion of very similar cells was almost entirely filtered by one method, thus suggesting that
potentially some cell types were exclusive only to the other method. This helped to dem-
onstrate the advantage of ddqc over a static threshold since it had many more unique
clusters than the static threshold method had. More detailed examples are provided in
the “Results” section.

Comparisons with miQC

At the time of testing, miQC was installed in R from GitHub using the command
“remotes::install_github("greenelab/miQC", build_vignettes = TRUE)”. miQC was run
on the test datasets (Tabula Muris heart and aorta and human olfactory epithelium)
using the standard steps as described in the vignette: https://​github.​com/​green​elab/​
miQC/​blob/​main/​vigne​ttes/​miQC.​Rmd. Comparison was performed by examining the
intersection of miQC retained barcodes with those retained by ddqc, leveraging the
annotations in the ddqc results.

Trends table (Additional file 1: Table S8)

We determined trends in QC metrics by iterating through all ddqc clusters in all tissues
and recording the clusters which satisfy one of the following criteria to a corresponding
table:

• Median number of genes lower than 200

• Median number of genes higher than 2000
• Median percent mito higher than 10
• Median percent ribo lower than 10
• Median percent ribo higher than 20

Comparison of clustering algorithms

In order to assess the performance of ddqc with different clustering algorithms, we have
used 4 algorithms provided by Pegasus (louvain, leiden, spectral louvain, and spectral lei-
den), and also implemented two additional algorithms: k-means and hierarchical cluster-
ing. For the algorithms within Pegasus, the clustering was performed using the function
pegasus.louvain, pegasus.leiden, pegasus.spectral_louvain, and pegasus.spectral_leiden
respectively. The k-means and hierarchical clustering methods were implemented using
sklearn.cluster.KMeans and sklearn.cluster.AgglomerativeClustering respectively. In both
algorithms, sklearn.metrics.silhouette_score was used to determine the number of clus-
ters. All functions were used with default parameters.
We ran ddqc using all 6 of those clustering algorithms for both initial and final cluster-
ing on Tabula Muris heart and aorta, and lung tissues. Then, we calculated the number
Subramanian et al. Genome Biology (2022) 23:267 Page 21 of 27

of cell barcodes that were retained by ddqc in the results of all six algorithms, as well as
the number of barcodes in pairwise intersections between different algorithms to deter-
mine if any one algorithm disproportionately retained more barcodes than the others.

Assessment of ddqc performance on the Seurat PBMC dataset

We ran ddqc on the PBMC dataset provided in the Seurat tutorial vignette (https://​satij​
alab.​org/​seurat/​artic​les/​get_​start​ed.​html). To get the clustering labels provided in the
tutorial, we repeated the tutorial steps in R and recorded the results into a csv file, clus-
ter labels from which were later used as cell annotations for the ddqc run on the same
data.
After comparing barcodes, we have identified that ddqc retains more cells than the
Seurat filter, and we have identified those cells and their celltypes. Also, we have looked
into logs provided by the ddqc to establish the cause of those cells being filtered out by
ddqc.

Comparison of ddqc with independent cell annotation methods

In order to assess the effectiveness of the graph-based clustering used in ddqc in parsing
out biological heterogeneity, we compared it with independent classification methods to
rule out any bias associated with clustering. We have used the following supervised and
unsupervised methods:

(1) SingleR [71]: https://​bioco​nduct​or.​org/​packa​ges/​relea​se/​bioc/​html/​Singl​eR.​html

(2) Azimuth [72]: https://​azimu​th.​hubma​pcons​ortium.​org/
(3) CellTypist [70]: https://​www.​cellt​ypist.​org/

We have performed a comparison on Seurat PBMC dataset using the steps below:

(1) First, we annotated each cell from the PBMC dataset provided in the Seurat tutorial
vignette (https://​satij​alab.​org/​seurat/​artic​les/​get_​start​ed.​html), following the steps
described in the respective method (SingleR, Azimuth, CellTypist)’s tutorial.
(2) Then, we mapped the annotation results with cell QC statistics and ddqc clustering
ID using the cell barcodes.
(3) Using the table function in R, we have calculated the intersections between ddqc
initial cluster IDs and automatic annotations (Additional file 1: Table S5)
(4) Finally, we did a run of ddqc on PBMC which used automatic annotations instead
of clustering. It was done similar to the original ddqc, excluding the clustering step
and grouping cells based on the independent annotation. Then the filtering cutoff
was calculated for each group using MAD with the threshold of 2. Filtering was
done on n_genes and percent_mito. We have compiled the results in Additional
file 1: Table S5:

a. ddqc_cluster: the initial clustering ID from original ddqc

b. single_r, azimuth, cell_typist: automatic annotation
Subramanian et al. Genome Biology (2022) 23:267 Page 22 of 27

c. %method_name%_passed_qc: whether the cell passed the QC based on a par-

ticular grouping method

All methods (including the independent annotation and the original graph-based clus-
tering using in ddqc) produced identical results.
We did a similar comparison for the Krasnow Lung dataset [47]. We only used Cell-
Typist as SingleR did not have a training dataset for Human Lung, and Azimuth’s web
interface had problems with processing this dataset. We evaluated results as for the
PBMC dataset described above.

Comparison of ddqc’s initial filtering for empty droplets and EmptyDrops

In order to assess robustness of the default Inital filtering in ddqc (cells with < 100
genes and > 80% mito are removed), we have compared it with EmptyDrops. We have
performed our comparison using the steps below:

(1) We downloaded the BAM and BAI files for Tabula Muris heart and lung dataset
from their S3 storage bucket (https://​s3.​conso​le.​aws.​amazon.​com/​s3/​bucke​ts/​czb-​
tabula-​muris-​senis?​prefix=​10x/3_​month/​&​region=​us-​west-2)
(2) We ran EmptyDrops for Tabula Muris heart and lung datasets using the DropletU-
tils [77] R package and followed the directions in the vignette.
(3) We have filtered out cells that had FDR more than 0.01 (as recommended in
theEmptyDrops vignette). We have compared the results of this filtering with ddqc’s
default by finding the number of common cells and cells that were retained by one
method and not the other.

Afterwards, we ran ddqc on the EmptyDrops filtering. We then compared its results
to regular ddqc with default filters by finding matches for each cluster among the
other method clusters similar to the approach described in “Comparison of ddqc with
independent cell classifiers section.”

Variable MAD muliplier analysis

We analyzed the performance of ddqc by running it with different thresholds by vary-
ing the MAD multiplier. We ran ddqc for each threshold from 1 to 3.5 with 0.1 incre-
ments and recorded the number and percentage of cells filtered out for each cluster.
We have also recorded other information, such as the cluster’s median, MAD, stand-
ard deviation, and MAD to SD ratio for n_counts, n_genes, and percent_mito.
Based on these results we have produced several plots:

• ggridges joyplot with rug broken down by cluster for each QC metric. Lines rep-
resenting 1 * MAD (red), 2 * MAD (green), and 3 * MAD (blue) for each cluster
were added to these plots in red, green, and blue colors respectively.
• Linechart with number or percentage of cells filtered on y-axis and threshold on
x-axis faceted by cluster. (this plot was also included in the released version of
Pegasus implementation of ddqc)
Subramanian et al. Genome Biology (2022) 23:267 Page 23 of 27

As part of this analysis, we have also predicted the modality of the distribution of
QC metrics for each cluster. For Tabula Muris heart and lung, we have run the follow-
ing functions:

• dip.test from diptest (https://​cran.r-​proje​ct.​org/​web/​packa​ges/​dipte​st/​index.​html)

• is.unimodal from LaplacesDemon (https://​cran.r-​proje​ct.​org/​web/​packa​ges/​L apla​
cesDe​mon/​index.​html)

For each cluster, we looked at n_counts, n_genes, and percent_mito and assessed
whether it was unimodal. We have considered the cluster unimodal if p_value was
less than 0.05 for diptest and if is.unimodal returned true for LaplacesDemon.

Visualization and plotting

Boxplots, joyplots, and violin plots for each QC metric were generated in R using the
ggplot2 and ggridges packages. For the tissue summary plots (Fig. 1), only initial or empty
droplet filtering was performed, and then the QC metrics plotted stratified by tissue.
For cell-type summary plots (Fig. 2), graph-based clustering was performed after initial
or empty droplet filtering. A horizontal red line for boxplots and violin plots, and verti-
cal line for joyplots were added to illustrate standard cutoff thresholds (10% for % mito-
chondrial transcripts, 200 for number of genes).
All analysis tasks were performed on the Broad Institute High-Performance Comput-
ing Cluster.

Supplementary Information
The online version contains supplementary material available at https://​doi.​org/​10.​1186/​s13059-​022-​02820-w.

Additional file 1: Tables S1-S9. Supplementary tables.

Additional file 2. Supplementary Text [15, 35, 38, 47, 70–72, 84–91].
Additional file 3. Supplementary figure legends and supplementary figures.
Additional file 4. Review history.

Acknowledgements
We are immensely grateful to Aviv Regev for her mentorship, helpful suggestions, and resources. We gratefully acknowl-
edge Oana Ursu, Sean Simmons, and Matan Hofree for critical review of the manuscript and Jan-Christian Huetter for
discussions. We acknowledge authors of several published studies who kindly responded to data requests or questions.
We thank Angela Pisco and James Webber (Tabula Muris), Kyle Joseph Travaglini (Human Lung dataset), Jonathan Man-
ning (EBI), and Dmitry Velmeshev (ASD snRNA-seq) for sharing datasets. We thank the MIT PRIMES program, the Broad
Institute Academic Affairs, Carrie E Wager, Elana Gonsalves, Anna Greka, Vijay Kuchroo, Ana Anderson, Rosy Hosking and
members of the Regev lab. We thank Leslie Gaffney for graphic design. Earlier iterations of this work were presented at
the Women in Statistics and Data Science Conference in 2018, and the MIT PRIMES Fall Conference in 2019.

Review history
The review history is available as Additional file 4.

Peer review information

Barbara Cheifet, Stephanie McClelland, and Veronique van den Berghe were the primary editors of this article and man-
aged its editorial process and peer review in collaboration with the rest of the editorial team.

Authors’ contributions
AS conceived the project, wrote the pilot version of ddqc, performed analysis, interpreted results, and wrote the paper.
MA co-developed ddqc with input from AS, performed analysis, interpreted results, and contributed to the “Methods”
section of the manuscript. YY and BL helped with integration of ddqc into Pegasus. All authors read the manuscript,
provided input, and have approved it for submission.
Subramanian et al. Genome Biology (2022) 23:267 Page 24 of 27

Authors’ information
Twitter handle: @ayshwaryas (Ayshwarya Subramanian); @malperovich1 (Mikhail Alperovich); @yang_yihming (Yiming
Yang); @BigGoodBo (Bo Li).

Funding
Not applicable.

Availability of data and materials

All datasets used in this manuscript are publicly available. Additional file 1: Tables S1 and S2 provide the information for
accessing the datasets. ddqc is available as a Python package on GitHub along with a tutorial: https://​github.​com/​ayshw​
aryas/​ddqc [78], and on Zenodo (https://​doi.​org/​10.​5281/​zenodo.​72972​80 [79]). For R users, a compatible package is
available on GitHub https://​github.​com/​ayshw​aryas/​ddqc_R [80] and on Zenodo (https://​doi.​org/​10.​5281/​zenodo.​72972​
76 [81]). The source code and all code used to generate the figures in the paper have been deposited on GitHub https://​
github.​com/​ayshw​aryas/​ddqc_​source [82] and on Zenodo (https://​doi.​org/​10.​5281/​zenodo.​72134​10 [83]). All code is
available for use under the open-source license BSD3.

Declarations
Ethics approval
Not applicable.

Competing interests
The authors declare that they have no competing interests.

Received: 18 August 2021 Accepted: 23 November 2022

References
1. Luecken MD, Theis FJ. Current best practices in single-cell RNA-seq analysis: a tutorial. Mol Syst Biol. 2019;15:e8746
EMBO.
2. Ilicic T, Kim JK, Kolodziejczyk AA, Bagger FO, McCarthy DJ, Marioni JC, et al. Classification of low quality cells from
single-cell RNA-seq data. Genome Biol. 2016;17:29.
3. Bach K, Pensa S, Grzelak M, Hadfield J, Adams DJ, Marioni JC, et al. Differentiation dynamics of mammary epithelial
cells revealed by single-cell RNA sequencing. Nat Commun. 2017;8:2128.
4. Young MD, Behjati S. SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing
data. Gigascience. 2020;9. https://​doi.​org/​10.​1093/​gigas​cience/​giaa1​51.
5. Fleming SJ, Marioni JC, Babadi M. CellBender remove-background: a deep generative model for unsupervised
removal of background noise from scRNA-seq datasets. Cold Spring Harbor Laboratory. 2019:791699 [cited 2021
Mar 4]. Available from: https://​www.​biorx​iv.​org/​conte​nt/​10.​1101/​79169​9v1.​abstr​act.
6. Yang S, Corbett SE, Koga Y, Wang Z, Johnson WE, Yajima M, et al. Decontamination of ambient RNA in single-cell
RNA-seq with DecontX. Genome Biol. 2020;21:57.
7. Lun ATL, Riesenfeld S, Andrews T, Dao TP, Gomes T, participants in the 1st Human Cell Atlas Jamboree, et al. Emp-
tyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data. Genome Biol.
2019;20:63.
8. Wolock SL, Lopez R, Klein AM. Scrublet: Computational Identification of cell doublets in single-cell transcriptomic
data. Cell Syst. 2019;8:281–291.e9.
9. McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using
artificial nearest neighbors. Cell Syst. 2019;8:329–337.e4.
10. A. Gayoso, J. Shor Doublet Detection Zenodo. 2018. https://​doi.​org/​10.​5281/​zenodo.​26780​42
11. Wolf FA, Angerer P, Theis FJ. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018;19:15.
12. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different condi-
tions, technologies, and species. Nat Biotechnol. 2018;36:411–20.
13. Mercer TR, Neph S, Dinger ME, Crawford J, Smith MA, Shearwood A-MJ, et al. The human mitochondrial transcrip-
tome. Cell. 2011;146:645–58.
14. Park J, Shrestha R, Qiu C, Kondo A, Huang S, Werth M, et al. Single-cell transcriptomics of the mouse kidney reveals
potential cellular targets of kidney disease. Science. 2018;360:758–63.
15. Kuppe C, Ibrahim MM, Kranz J, Zhang X, Ziegler S, Perales-Patón J, et al. Decoding myofibroblast origins in human
kidney fibrosis. Nature. 2021;589:281–6.
16. Bortoluzzi S, d’Alessi F, Romualdi C, Danieli GA. Differential expression of genes coding for ribosomal proteins in dif-
ferent human tissues. Bioinformatics. 2001;17:1152–7.
17. Kondrashov N, Pusic A, Stumpf CR, Shimizu K, Hsieh AC, Ishijima J, et al. Ribosome-mediated specificity in Hox
mRNA translation and vertebrate tissue patterning. Cell. 2011;145:383–97.
18. Thorrez L, Van Deun K, Tranchevent L-C, Van Lommel L, Engelen K, Marchal K, et al. Using ribosomal protein genes as
reference: a tale of caution. PLoS One. 2008;3:e1854.
19. Gulati GS, Sikandar SS, Wesche DJ, Manjunath A, Bharadwaj A, Berger MJ, et al. Single-cell transcriptional diversity is
a hallmark of developmental potential. Science. 2020;367:405–11.
20. Lin J, Amir A. Homeostasis of protein and mRNA concentrations in growing cells. Nat Commun. 2018;9:4496.
Subramanian et al. Genome Biology (2022) 23:267 Page 25 of 27

21. Padovan-Merhar O, Nair GP, Biaesch AG, Mayer A, Scarfone S, Foley SW, et al. Single mammalian cells compensate for
differences in cellular volume and DNA copy number through independent global transcriptional mechanisms. Mol
Cell. 2015;58:339–52.
22. Asmal M, Colgan J, Naef F, Yu B, Lee Y, Magnasco M, et al. Production of ribosome components in effector CD4+ T
cells is accelerated by TCR stimulation and coordinated by ERK-MAPK. Immunity. 2003;19:535–48.
23. Ricciardi S, Manfrini N, Alfieri R, Calamita P, Crosti MC, Gallo S, et al. The translational machinery of human CD4+
T cells is poised for activation and controls the switch from quiescence to metabolic remodeling. Cell Metab.
2018;28:961.
24. Pogue-Geile K, Geiser JR, Shu M, Miller C, Wool IG, Meisler AI, et al. Ribosomal protein genes are overexpressed
in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein. Mol Cell Biol.
1991;11:3842–9.
25. Wallrapp A, Riesenfeld SJ, Burkett PR, Abdulnour R-EE, Nyman J, Dionne D, et al. Erratum: the neuropeptide NMU
amplifies ILC2-driven allergic lung inflammation. Nature. 2017;551:658.
26. Osorio D, Cai JJ. Systematic determination of the mitochondrial proportion in human and mice tissues for single-
cell RNA-sequencing data quality control. Bioinformatics. 2020. https://​doi.​org/​10.​1093/​bioin​forma​tics/​btaa7​51.
27. Slyper M, Porter CBM, Ashenberg O, Waldman J, Drokhlyansky E, Wakiro I, et al. Author Correction: a single-cell and
single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat Med. 2020;26:1307.
28. McCarthy DJ, Campbell KR, Lun ATL, Wills QF. Scater: pre-processing, quality control, normalization and visualization of
single-cell RNA-seq data in R. Bioinformatics. 2017;33:1179–86.
29. Hippen AA, Falco MM, Weber LM, Erkan EP, Zhang K, Doherty JA, et al. miQC: An adaptive probabilistic framework for
quality control of single-cell RNA-sequencing data. PLoS Comput Biol. 2021;17(8):e1009290. https://​doi.​org/​10.​1371/​
journ​al.​pcbi.​10092​90. PMID: 34428202; PMCID: PMC8415599
30. Muus C, Luecken MD, Eraslan G, Sikkema L, Waghray A, Heimberg G, et al. Single-cell meta-analysis of SARS-CoV-2 entry
genes across tissues and demographics. Nat Med. 2021;27:546–59.
31. Breton G, Zheng S, Valieris R, Tojal da Silva I, Satija R, Nussenzweig MC. Human dendritic cells (DCs) are derived from
distinct circulating precursors that are precommitted to become CD1c+ or CD141+ DCs. J Exp Med. 2016;213:2861–70
Rockefeller University Press.
32. Wu Z, Gao S, Zhao X, Chen J, Keyvanfar K, Feng X, et al. Long noncoding RNAs of single hematopoietic stem and pro-
genitor cells in healthy and dysplastic human bone marrow. Haematologica. 2019;104:894–906 Ferrata Storti Founda-
tion (Haematologica).
33. Nguyen QH, Pervolarakis N, Blake K, Ma D, Davis RT, James N, et al. Profiling human breast epithelial cells using single cell
RNA sequencing identifies cell diversity. Nat Commun. 2018;9. https://​doi.​org/​10.​1038/​s41467-​018-​04334-1 Springer
Science and Business Media LLC.
34. Wang YJ, Schug J, Won K-J, Liu C, Naji A, Avrahami D, et al. Single-cell transcriptomics of the human endocrine pancreas.
Diabetes. 2016;65:3028–38.
35. Vieira Braga FA, Kar G, Berg M, Carpaij OA, Polanski K, Simon LM, et al. A cellular census of human lungs identifies novel
cell states in health and in asthma. Nat Med. 2019;25:1153–63.
36. Xu Y, Mizuno T, Sridharan A, Du Y, Guo M, Tang J, et al. Single-cell RNA sequencing identifies diverse roles of epithelial
cells in idiopathic pulmonary fibrosis. JCI Insight. 2016;1:e90558.
37. Lambrechts D, Wauters E, Boeckx B, Aibar S, Nittner D, Burton O, et al. Phenotype molding of stromal cells in the lung
tumor microenvironment. Nat Med. 2018;24:1277–89 Springer Science and Business Media LLC.
38. Enge M, Efsun Arda H, Mignardi M, Beausang J, Bottino R, Kim SK, et al. Single-cell analysis of human pancreas reveals
transcriptional signatures of aging and somatic mutation patterns. Cell. 2017:321–330.e14. https://​doi.​org/​10.​1016/j.​cell.​
2017.​09.​004.
39. Lawlor N, George J, Bolisetty M, Kursawe R, Sun L, Sivakamasundari V, et al. Single-cell transcriptomes identify human
islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes. Genome Res. 2017;27:208–22
Cold Spring Harbor Laboratory.
40. Xin Y, Kim J, Okamoto H, Ni M, Wei Y, Adler C, et al. RNA sequencing of single human islet cells reveals type 2 diabetes
genes. Cell Metab. 2016;24:608–15 Elsevier BV.
41. Lukowski SW, Lo CY, Sharov AA, Nguyen Q, Fang L, Hung SS, et al. A single-cell transcriptome atlas of the adult human
retina. EMBO J. 2019;38:e100811 EMBO.
42. Han X, Zhou Z, Fei L, Sun H, Wang R, Chen Y, et al. Construction of a human cell landscape at single-cell level. Nature.
2020;581:303–9.
43. He J, Lin Y, Meng M, Li J, Yang JY, Wang H. Construction of a human cell landscape of COVID-19 infection at single-cell
level. Aging Dis. 2021;12:705–9.
44. Eraslan G, Drokhlyansky E, Anand S, Fiskin E, Subramanian A, Slyper M, et al. Single-nucleus cross-tissue molecular refer-
ence maps toward understanding disease gene function. Science. 2022;376:eabl4290.
45. Peng Y-R, Shekhar K, Yan W, Herrmann D, Sappington A, Bryman GS, et al. Molecular classification and comparative
taxonomics of foveal and peripheral cells in primate retina. Cell. 2019;176:1222–1237.e22.
46. Durante MA, Kurtenbach S, Sargi ZB, Harbour JW, Choi R, Kurtenbach S, et al. Single-cell analysis of olfactory neurogen-
esis and differentiation in adult humans. Nat Neurosci. 2020;23:323–6 Springer Science and Business Media LLC.
47. Travaglini KJ, Nabhan AN, Penland L, Sinha R, Gillich A, Sit RV, et al. A molecular cell atlas of the human lung from single-
cell RNA sequencing. Nature. 2020;587:619–25.
48. Andrews TS, Atif J, Liu JC, Perciani CT, Ma X-Z, Thoeni C, et al. Single-cell, single-nucleus, and spatial RNA sequencing of
the human liver identifies cholangiocyte and mesenchymal heterogeneity. Hepatol Commun. 2022;6:821–40 Wiley.
49. Liao J, Yu Z, Chen Y, Bao M, Zou C, Zhang H, et al. Single-cell RNA sequencing of human kidney. Sci Data. 2020;7:4
Springer Science and Business Media LLC.
50. Tucker NR, Chaffin M, Fleming SJ, Hall AW, Parsons VA, Bedi KC Jr, et al. Transcriptional and cellular diversity of the human
heart. Circulation. 2020;142:466–82.
51. Smillie CS, Biton M, Ordovas-Montanes J, Sullivan KM, Burgin G, Graham DB, et al. Intra- and inter-cellular rewiring of the
human colon during ulcerative colitis. Cell. 2019;178:714–f Elsevier BV.
Subramanian et al. Genome Biology (2022) 23:267 Page 26 of 27

52. Gaublomme JT, Li B, McCabe C, Knecht A, Yang Y, Drokhlyansky E, et al. Nuclei multiplexing with barcoded antibodies for
single-nucleus genomics. Nat Commun. 2019;10:2907 Springer Science and Business Media LLC.
53. Velmeshev D, Schirmer L, Jung D, Haeussler M, Perez Y, Mayer S, et al. Single-cell genomics identifies cell type-specific
molecular changes in autism. Science. 2019;364:685–9 American Association for the Advancement of Science (AAAS).
54. Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, et al. Single-cell reconstruction of the early
maternal-fetal interface in humans. Nature. 2018;563:347–53 Springer Science and Business Media LLC.
55. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, et al. Highly Parallel genome-wide expression profiling
of individual cells using nanoliter droplets. Cell. 2015;161:1202–14.
56. Tabula Muris Consortium, Overall coordination, Logistical coordination, Organ collection and processing, Library
preparation and sequencing, Computational data analysis, et al. Single-cell transcriptomics of 20 mouse organs creates
a Tabula Muris. Nature. 2018;562:367–72.
57. Han X, Wang R, Zhou Y, Fei L, Sun H, Lai S, et al. Mapping the mouse cell atlas by microwell-seq. Cell. 2018;173:1307
Elsevier BV.
58. Tabula Muris Consortium. A single-cell transcriptomic atlas characterizes ageing tissues in the mouse. Nature.
2020;583:590–5 Springer Science and Business Media LLC.
59. Franzén O, Gan L-M, Björkegren JLM. PanglaoDB: a web server for exploration of mouse and human single-cell RNA
sequencing data. Database. 2019;2019. https://​doi.​org/​10.​1093/​datab​ase/​baz046.
60. Sohni A, Tan K, Song H-W, Burow D, de Rooij DG, Laurent L, et al. The neonatal and adult human testis defined at the
single-cell level. Cell Rep. 2019;26:1501–1517.e4 Elsevier BV.
61. Henry GH, Malewska A, Joseph DB, Malladi VS, Lee J, Torrealba J, et al. A cellular anatomy of the normal adult human
prostate and prostatic urethra. Cell Rep. 2018;25:3530–3542.e5 Elsevier BV.
62. Xin Y, Dominguez Gutierrez G, Okamoto H, Kim J, Lee A-H, Adler C, et al. Pseudotime ordering of single human β-cells
reveals states of insulin production and unfolded protein response. Diabetes. 2018;67:1783–94.
63. Merino D, Weber TS, Serrano A, Vaillant F, Liu K, Pal B, et al. Barcoding reveals complex clonal behavior in patient-derived
xenografts of metastatic triple negative breast cancer. Nat Commun. 2019:10. https://​doi.​org/​10.​1038/​s41467-​019-​
08595-2 Springer Science and Business Media LLC.
64. Habiel DM, Espindola MS, Jones IC, Coelho AL, Stripp B, Hogaboam CM. CCR10+ epithelial cells from idiopathic pulmo-
nary fibrosis lungs drive remodeling. JCI Insight. 2018;3. https://​doi.​org/​10.​1172/​jci.​insig​ht.​122211.
65. Welch JD, Kozareva V, Ferreira A, Vanderburg C, Martin C, Macosko EZ. Single-cell multi-omic integration compares and
contrasts features of brain cell identity. Cell. 2019;177:1873–1887.e17 Elsevier BV.
66. Oetjen KA, Lindblad KE, Goswami M, Gui G, Dagur PK, Lai C, et al. Human bone marrow assessment by single-cell RNA
sequencing, mass cytometry, and flow cytometry. JCI Insight. 2018:3. https://​doi.​org/​10.​1172/​jci.​insig​ht.​124928 Ameri-
can Society for Clinical Investigation.
67. MacParland SA, Liu JC, Ma X-Z, Innes BT, Bartczak AM, Gage BK, et al. Single cell RNA sequencing of human liver reveals
distinct intrahepatic macrophage populations. Nat Commun. 2018;9:4383 Springer Science and Business Media LLC.
68. Shekhar K, Lapan SW, Whitney IE, Tran NM, Macosko EZ, Kowalczyk M, et al. Comprehensive classification of retinal
bipolar neurons by single-cell transcriptomics. Cell. 2016;166:1308–1323.e30.
69. Li B, Gould J, Yang Y, Sarkizova S, Tabaka M, Ashenberg O, et al. Cumulus provides cloud-based data analysis for large-
scale single-cell and single-nucleus RNA-seq. Nat Methods. 2020;17:793–8.
70. Domínguez Conde C, Xu C, Jarvis LB, Rainbow DB, Wells SB, Gomes T, et al. Cross-tissue immune cell analysis reveals
tissue-specific features in humans. Science. 2022;376:eabl5197.
71. Aran D, Looney AP, Liu L, Wu E, Fong V, Hsu A, et al. Reference-based analysis of lung single-cell sequencing reveals a
transitional profibrotic macrophage. Nat Immunol. 2019;20:163–72.
72. Hao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S, Butler A, et al. Integrated analysis of multimodal single-cell
data. Cell. 2021;184:3573–3587.e29.
73. Wolf T, Jin W, Zoppi G, Vogel IA, Akhmedov M, Bleck CKE, et al. Dynamics in protein translation sustaining T cell prepared-
ness. Nat Immunol. 2020;21:927–37.
74. Svensson V, da Veiga BE, Pachter L. A curated database reveals trends in single-cell transcriptomics. Database. 2020;2020.
https://​doi.​org/​10.​1093/​datab​ase/​baaa0​73.
75. Malkov YA, Yashunin DA. Efficient and robust approximate nearest neighbor search using hierarchical Navigable Small
World graphs. IEEE Trans Pattern Anal Mach Intell. 2020;42:824–36 Institute of Electrical and Electronics Engineers (IEEE).
76. Blondel VD, Guillaume J-L, Lambiotte R, Lefebvre E. Fast unfolding of communities in large networks. J Stat Mech: Theory
Exp. 2008:P10008. https://​doi.​org/​10.​1088/​1742-​5468/​2008/​10/​p10008.
77. Lun ATL, Riesenfeld S, Andrews T, Dao T, Gomes T, participants in the 1st Human Cell Atlas Jamboree, Marioni JC.
EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data. Genome Biol.
2019;20:63. https://​doi.​org/​10.​1186/​s13059-​019-​1662-y.
78. Subramanian A, Alperovich M, Yang Y, Li B. Biology-inspired data-driven quality control for scientific discovery in single-
cell transcriptomics: GitHub; 2022. https://​github.​com/​ayshw​aryas/​ddqc
79. Subramanian A, Alperovich M, Yang Y, Li B. Biology-inspired data-driven quality control for scientific discovery in single-
cell transcriptomics: Zenodo; 2022. https://​doi.​org/​10.​5281/​zenodo.​72972​80.
80. Subramanian A, Alperovich M, Yang Y, Li B. Biology-inspired data-driven quality control for scientific discovery in single-
cell transcriptomics: GitHub; 2022. https://​github.​com/​ayshw​aryas/​ddqc_R
81. Subramanian A, Alperovich M, Yang Y, Li B. Biology-inspired data-driven quality control for scientific discovery in single-
cell transcriptomics: Zenodo; 2022. https://​doi.​org/​10.​5281/​zenodo.​72972​76.
82. Subramanian A, Alperovich M, Yang Y, Li B. Biology-inspired data-driven quality control for scientific discovery in single-
cell transcriptomics: GitHub; 2022. https://​github.​com/​ayshw​aryas/​ddqc_​source
83. Subramanian A, Alperovich M, Yang Y, Li B. Biology-inspired data-driven quality control for scientific discovery in single-
cell transcriptomics: Zenodo; 2022. https://​doi.​org/​10.​5281/​zenodo.​72134​10.
84. Slyper M, Porter CBM, Ashenberg O, Waldman J, Drokhlyansky E, Wakiro I, et al. A single-cell and single-nucleus RNA-Seq
toolbox for fresh and frozen human tumors. Nat Med. 2020;26:792–802 Springer Science and Business Media LLC.
Subramanian et al. Genome Biology (2022) 23:267 Page 27 of 27

85. Nowakowski TJ, Bhaduri A, Pollen AA, Alvarado B, Mostajo-Radji MA, Di Lullo E, et al. Spatiotemporal gene expression
trajectories reveal developmental hierarchies of the human cortex. Science. 2017;358:1318–23.
86. Szabo PA, Levitin HM, Miron M, Snyder ME, Senda T, Yuan J, et al. Single-cell transcriptomics of human T cells reveals
tissue and activation signatures in health and disease. Nat Commun. 2019;10:4706.
87. Qiu S, Hong R, Zhuang Z, Wang S. Abstract 1763A: Single-cell RNA-sequencing reveals the immune contexture of triple-
negative breast cancer tumors. Tumor Biol. 2018. https://​doi.​org/​10.​1158/​1538-​7445.​am2018-​1763a.
88. Aizarani N, Saviano A, Mailly L, Durand S, Herman JS, et al. A human liver cell atlas reveals heterogeneity and epithelial
progenitors. Nature. 2019;572:199–204 Springer Science and Business Media LLC.
89. Mucenski ML, Mahoney R, Adam M, Potter AS, Potter SS. Single cell RNA-seq study of wild type and Hox9,10,11 mutant
developing uterus. Sci Rep. 2019;9:4557.
90. Kim D, Kobayashi T, Voisin B, Jo J-H, Sakamoto K, Jin S-P, et al. Targeted therapy guided by single-cell transcriptomic
analysis in drug-induced hypersensitivity syndrome: a case report. Nat Med. 2020;26:236–43.
91. Slevin SM, Garner LC, Lahiff C, Tan M, Wang LM, Ferry H, et al. Lymphocyte activation gene (LAG)-3 is associated with
mucosal inflammation and disease activity in ulcerative colitis. J Crohns Colitis. 2020;14:1446–61.

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