11.8 通路水平基因表达分析（单细胞下游分析ReactomeGSA包学习）

一.细胞簇通路水平基因表达包学习

Analysing single-cell RNA-sequencing Data

ReactomeGSA包是基于web的Reactome分析系统的客户端。本质上，它使用最新版本的Reactome通路数据库作为后端进行基因集分析。

 “The pathway analysis is at the very end of a scRNA-seq workflow. This means, that any Q/C was already performed, the data was normalized and cells were already clustered.

The ReactomeGSA package can now be used to get pathway-level expression values for every cell cluster. This is achieved by calculating the mean gene expression for every cluster and then submitting this data to a gene set variation analysis.

All of this is wrapped in the single analyse_sc_clusters function.”

通路分析是单细胞分析较后的步骤，此时Q/C已经执行，数据已经标准化细胞已经聚类

这个包是用来分析每一个细胞簇通路水平的基因表达，方法是算每一簇基因表达平均值，然后把这些数据上传进行基因集差异分析，返回的对象是：

“pathways returns the pathway-level expression values per cell cluster:”

pathway函数返回值为每一细胞簇通路水平基因表达

“A simple approach to find the most relevant pathways is to assess the maximum difference in expression for every pathway”

单可视化：

多可视化：

二、T-cell exhaustion signatures characterize the immune landscape and predict HCC文献泛读

文章联合单细胞测序和bulk rna-seq，寻找与T细胞耗竭相关的biomarker

重点关注了单细胞数据分析：seurat经典流程，细胞注释用singleR,热图展示marker基因在细胞簇表达，细胞类型功能分析用的是通路水平的The ReactomeGSA package,然后做了Cellular communication networks， were inferred by calculating the likelihood of communication. Intercellular communication network studies show that “HLA-DPA 1-CD 4 plays an important role in the intercellular communication network”. 

关于单细胞处理作者在discussion中的描述：“We obtained T-cell maker genes by dimensionality reduction and clustering based on scRNA-seq data from the GSE166635 dataset. ”

三、Circulating tumor cell characterization of lung cancer brain metastases in the cerebrospinal fluid through single‐cell transcriptome analysis

通过单细胞转录组分析肺癌脑转移脑脊液中循环肿瘤细胞的特征

通过Smart‐seq2单细胞RNA测序（scRNA‐seq）研究了CSF‐CTC的转录组特征。我们招募了5名LUAD‐LM患者，调查了1000多个CSF‐CTC的转录谱。通过在单细胞水平上分析LMs患者的CSF‐CTC的转录组特征，可以首次揭示CSF‐CTC的肿瘤内和肿瘤间异质性。此外，CSF‐CTC特征基因的发现可用于结合基于RNA的分子标记，以进一步进行临床诊断，并促进在应对LMs临床挑战方面的潜在突破。

流式细胞分离CTC,normal 和patient做单细胞测序，细胞聚类病人组和对照组分开，病人组除了某一个病人的12个单核细胞离群外其余都是病人聚病人

 The ImmuneScore was computed based on Estimation of STromal and Immune cells in MAlignant Tumors using Expression data (ESTIMATE) R package.

细胞聚类：To infer the cell type identity, Seurat 3.0 was used to generate expression heatmaps of selected gene markers of known cell types, including T cells (CD2,CD3D,CD3E, andCD3G), B cells (CD19,MS4A1,CD79A, andCD79B), monocytes (CD14,CD68, andCD163), lung cells (SFTPA1,SFTPA2,SFTPB, andNAPSA), epithelial cells (EPCAM,CDH1,KRT7,KRT8,KRT18, andMUC1), and proliferation cells (CCND1andTOP2A). 使用选择的基因marker绘制热图

差异表达分析和通路富集分析

ignificantly differentially expressed genes (DEGs) between samples were detected by DESeq224using normalized gene expression counts, at an adjustedP‐value cutoff of .05 and a fold‐change cutoff of 2. Gene set enrichment analysis (GSEA) was used for functional enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways.对照组和患者组差异基因分析，用的是标准化的基因表达count(应该就是RNA assay里面的data)。

Cell cycle analysis（细胞周期分析）： using the CellCycleScoring function included in Seurat 3.0 package。

四，预备工作

单细胞取亚群，做mean expression(已放到文件收藏夹)

不同时间段不同假基因表达变化分析

继续阅读文献看其他人的分析方法，这样做了能干什么？——disturbing problems

最近英语好烂，改提升一下（想做什么千万别等，就直接做，错了就错了呗）