Seurat单细胞测序

Seurat 4.0 || WNN整合scRNA和scATAC数

2020-10-31  本文已影响0人  周运来就是我

前情回顾

Seurat 4.0 ||单细胞数据分析工具箱有更新
Seurat 4.0 ||单细胞多模态数据整合算法WNN
Seurat 4.0 || 分析scRNA和表面抗体数据

单细胞数据数据正在走向统一:在一个细胞内同时测量分属中心法则不同阶段的多种数据类型。这需要我们开发新的方法来将这些数据整合在一起以描绘一个完整的细胞状态。Seurat升级到4.0以后,在一个Seurat对象中可以存储(数据结构)和计算(算法)单细胞多模态数据。本文我们跟着官方教程演示使用WNN分析多模态技术分析10xscRNA+ATAC数据。使用的数据集在10x网站上公开,是为10412个同时测量转录组和ATAC的PBMCs细胞。

在这个例子中,我们将演示:

你可以从10x dataset网站下载数据集。请务必下载以下文件:

Filtered feature barcode matrix (HDF5)
ATAC Per fragment information file (TSV.GZ)
ATAC Per fragment information index (TSV.GZ index) # 这个索引文件别忘了哦
网址:https://support.10xgenomics.com/single-cell-multiome-atac-gex/datasets/1.0.0/pbmc_granulocyte_sorted_10k?

最后,为了运行本示例,请确保安装了以下软件包:

library(Seurat)
library(Signac)
library(EnsDb.Hsapiens.v86)
library(dplyr)
library(ggplot2)

我们将基于基因表达数据创建一个Seurat对象,然后添加ATAC-seq数据作为第二个assay。关于创建和处理ATAC对象的更多信息,可以浏览Signac相关文档。

# the 10x hdf5 file contains both data types. 
inputdata.10x <- Read10X_h5("../data/pbmc_granulocyte_sorted_10k_filtered_feature_bc_matrix.h5")

# extract RNA and ATAC data
rna_counts <- inputdata.10x$`Gene Expression`
atac_counts <- inputdata.10x$Peaks

# Create Seurat object
pbmc <- CreateSeuratObject(counts = rna_counts)
pbmc[["percent.mt"]] <- PercentageFeatureSet(pbmc, pattern = "^MT-")

head(pbmc@meta.data)
                      orig.ident nCount_RNA nFeature_RNA percent.mt
AAACAGCCAAGGAATC-1 SeuratProject       8380         3308   7.470167
AAACAGCCAATCCCTT-1 SeuratProject       3771         1896  10.527711
AAACAGCCAATGCGCT-1 SeuratProject       6876         2904   6.457243
AAACAGCCACACTAAT-1 SeuratProject       1733          846  18.003462
AAACAGCCACCAACCG-1 SeuratProject       5415         2282   6.500462
AAACAGCCAGGATAAC-1 SeuratProject       2759         1353   6.922798

VlnPlot(pbmc,features=c('percent.mt','nCount_RNA', 'nFeature_RNA'),pt.size = 0,cols = "red")

添加ATAC数据。


# Now add in the ATAC-seq data
# we'll only use peaks in standard chromosomes
grange.counts <- StringToGRanges(rownames(atac_counts), sep = c(":", "-"))
grange.use <- seqnames(grange.counts) %in% standardChromosomes(grange.counts)
atac_counts <- atac_counts[as.vector(grange.use), ]
annotations <- GetGRangesFromEnsDb(ensdb = EnsDb.Hsapiens.v86)
seqlevelsStyle(annotations) <- 'UCSC'
genome(annotations) <- "hg38"
frag.file <- "/home/data/vip06/dataset/pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz"



chrom_assay <- CreateChromatinAssay(
  counts = atac_counts,
  sep = c(":", "-"),
  genome = 'hg38',
  fragments = frag.file,
  min.cells = 10,
  annotation = annotations
)
pbmc[["ATAC"]] <- chrom_assay

查看数据集对象:


pbmc[["ATAC"]]
ChromatinAssay data with 105913 features for 11909 cells
Variable features: 0 
Genome: hg38 
Annotation present: TRUE 
Motifs present: FALSE 
Fragment files: 1 


pbmc
An object of class Seurat 
142514 features across 11909 samples within 2 assays 
Active assay: RNA (36601 features, 0 variable features)
 1 other assay present: ATAC

我们根据每种模式检测到的分子数以及线粒体百分比来执行基本的QC,其实这一步什么也没有Q掉,只是获得数据概览,了解数据的质量分布。

 head(pbmc@meta.data)
                      orig.ident nCount_RNA nFeature_RNA percent.mt nCount_ATAC nFeature_ATAC
AAACAGCCAAGGAATC-1 SeuratProject       8380         3308   7.470167       55550         13867
AAACAGCCAATCCCTT-1 SeuratProject       3771         1896  10.527711       20485          7247
AAACAGCCAATGCGCT-1 SeuratProject       6876         2904   6.457243       16674          6528
AAACAGCCACACTAAT-1 SeuratProject       1733          846  18.003462        2007           906
AAACAGCCACCAACCG-1 SeuratProject       5415         2282   6.500462        7658          3323
AAACAGCCAGGATAAC-1 SeuratProject       2759         1353   6.922798       10355          4267


VlnPlot(pbmc, features = c("nCount_RNA", "nFeature_RNA", "percent.mt", "nCount_ATAC", "nFeature_ATAC"), ncol = 3,
        log = TRUE, pt.size = 0) + NoLegend()


如果是多个样本这样的展示方式可以看到每个样本的质量差异,咦,WNN框架下如何执行多样本分析?这里我们还是假设有那么一组吧,人为设置一个分组。

pbmc$replicate <- sample(c("ctrl", "para"), size = ncol(pbmc), replace = TRUE)
VlnPlot(pbmc, features = c("nCount_RNA", "nFeature_RNA", "percent.mt", "nCount_ATAC", "nFeature_ATAC"), ncol = 3,group.by = 'replicate',
        log = TRUE, pt.size = 0) + NoLegend()

看到数据的质量分布之后,我们可以执行真正的质控了,把异常值或者不是细胞的barcode去掉,保证进入分析的都是细胞,而非杂质。

pbmc <- subset(
  x = pbmc,
  subset = nCount_ATAC < 7e4 &
    nCount_ATAC > 5e3 &
    nCount_RNA < 25000 &
    nCount_RNA > 1000 &
    percent.mt < 20
)

接下来,我们使用RNA和ATAC-seq数据的标准方法,分别对这两种检测方法进行预处理和降维。

# RNA analysis
DefaultAssay(pbmc) <- "RNA"
pbmc <- SCTransform(pbmc, verbose = FALSE) %>% RunPCA() %>% RunUMAP(dims = 1:50, reduction.name = 'umap.rna', reduction.key = 'rnaUMAP_')
PC_ 1 
Positive:  VCAN, PLXDC2, SAT1, SLC8A1, NAMPT, NEAT1, ZEB2, DPYD, LRMDA, LYZ 
       FCN1, LYN, ANXA1, JAK2, CD36, AOAH, ARHGAP26, TYMP, RBM47, PLCB1 
       PID1, LRRK2, ACSL1, LYST, IRAK3, DMXL2, MCTP1, AC020916.1, GAB2, TBXAS1 
Negative:  EEF1A1, RPL13, BCL2, RPS27, RPL13A, LEF1, BCL11B, BACH2, INPP4B, RPL41 
       IL32, RPS27A, CAMK4, IL7R, RPL3, SKAP1, RPS2, GNLY, LTB, CD247 
       RPS12, RPS18, RPL11, PRKCH, ANK3, RPL23A, THEMIS, CCL5, RPL10, RPS3 
PC_ 2 
Positive:  BANK1, IGHM, AFF3, IGKC, RALGPS2, MS4A1, PAX5, CD74, EBF1, FCRL1 
       CD79A, OSBPL10, LINC00926, COL19A1, BLK, NIBAN3, IGHD, CD22, HLA-DRA, ADAM28 
       CD79B, PLEKHG1, COBLL1, AP002075.1, LIX1-AS1, CCSER1, TCF4, BCL11A, GNG7, STEAP1B 
Negative:  GNLY, CCL5, NKG7, CD247, IL32, PRKCH, PRF1, BCL11B, INPP4B, LEF1 
       IL7R, CAMK4, THEMIS, GZMA, RORA, GZMH, TXK, TC2N, TRBC1, PITPNC1 
       SYNE2, PDE3B, STAT4, TRAC, TGFBR3, VCAN, KLRD1, NCALD, CTSW, SLFN12L 
PC_ 3 
Positive:  LEF1, CAMK4, INPP4B, PDE3B, IL7R, FHIT, BACH2, MAML2, TSHZ2, SERINC5 
       BCL2, EEF1A1, NELL2, ANK3, CCR7, PRKCA, TCF7, BCL11B, LTB, AC139720.1 
       OXNAD1, MLLT3, RPL11, AL589693.1, TRABD2A, RPL13, RASGRF2, MAL, NR3C2, LDHB 
Negative:  GNLY, NKG7, CCL5, PRF1, GZMA, GZMH, KLRD1, SPON2, FGFBP2, CST7 
       GZMB, CCL4, TGFBR3, KLRF1, CTSW, BNC2, ADGRG1, PDGFD, PPP2R2B, PYHIN1 
       IL2RB, CLIC3, A2M, NCAM1, MCTP2, TRDC, C1orf21, SAMD3, MYBL1, FCRL6 
PC_ 4 
Positive:  TCF4, CUX2, AC023590.1, LINC01374, RHEX, LINC01478, EPHB1, PLD4, PTPRS, ZFAT 
       LINC00996, CLEC4C, LILRA4, COL26A1, PLXNA4, SCN9A, CCDC50, FAM160A1, ITM2C, COL24A1 
       UGCG, RUNX2, GZMB, NRP1, PHEX, IRF8, JCHAIN, SLC35F3, PACSIN1, AC007381.1 
Negative:  BANK1, IGHM, MS4A1, PAX5, IGKC, FCRL1, EBF1, RALGPS2, CD79A, LINC00926 
       OSBPL10, GNLY, COL19A1, IGHD, NKG7, CD22, BACH2, CCL5, PLEKHG1, CD79B 
       ADAM28, LIX1-AS1, LARGE1, STEAP1B, AP002075.1, AC120193.1, BLK, FCER2, ARHGAP24, IFNG-AS1 
PC_ 5 
Positive:  VCAN, PLCB1, ANXA1, PLXDC2, DPYD, CD36, ARHGAP26, ARHGAP24, ACSL1, AC020916.1 
       LRMDA, DYSF, CSF3R, MEGF9, PDE4D, S100A8, FNDC3B, ADAMTSL4-AS1, S100A9, TMTC2 
       RAB11FIP1, CREB5, LRRK2, NAMPT, RBM47, JUN, SLC2A3, GNLY, GLT1D1, VCAN-AS1 
Negative:  CDKN1C, FCGR3A, TCF7L2, IFITM3, CST3, MTSS1, AIF1, LST1, MS4A7, PSAP 
       WARS, ACTB, HLA-DPA1, SERPINA1, CD74, FCER1G, IFI30, COTL1, CFD, SMIM25 
       HLA-DRA, HLA-DRB1, FMNL2, CSF1R, FTL, MAFB, TMSB4X, TYROBP, HLA-DPB1, CCDC26 
06:10:45 UMAP embedding parameters a = 0.9922 b = 1.112
06:10:45 Read 10412 rows and found 50 numeric columns
06:10:45 Using Annoy for neighbor search, n_neighbors = 30
06:10:45 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
06:10:46 Writing NN index file to temp file /tmp/Rtmp3eAPNF/file3dd9069c738d7
06:10:46 Searching Annoy index using 1 thread, search_k = 3000
06:10:49 Annoy recall = 100%
06:10:50 Commencing smooth kNN distance calibration using 1 thread
06:10:52 Initializing from normalized Laplacian + noise
06:10:53 Commencing optimization for 200 epochs, with 443480 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
06:10:59 Optimization finished
There were 50 or more warnings (use warnings() to see the first 50)

分析ATAC数据,ATAC数据在这里也是一个矩阵,只是不是RNA含量而是fragments矩阵。但是ATAC的assays更丰富。

 str(pbmc@assays$ATAC,max.level = 2)
Formal class 'ChromatinAssay' [package "Signac"] with 16 slots
  ..@ ranges            :Formal class 'GRanges' [package "GenomicRanges"] with 7 slots
  ..@ motifs            : NULL
  ..@ fragments         :List of 1
  ..@ seqinfo           :Formal class 'Seqinfo' [package "GenomeInfoDb"] with 4 slots
  ..@ annotation        :Formal class 'GRanges' [package "GenomicRanges"] with 7 slots
  ..@ bias              : NULL
  ..@ positionEnrichment: list()
  ..@ links             :Formal class 'GRanges' [package "GenomicRanges"] with 7 slots
  ..@ counts            :Formal class 'dgCMatrix' [package "Matrix"] with 6 slots
  ..@ data              :Formal class 'dgCMatrix' [package "Matrix"] with 6 slots
  ..@ scale.data        : num[0 , 0 ] 
  ..@ key               : chr "atac_"
  ..@ assay.orig        : NULL
  ..@ var.features      : chr [1:105913] "chr19-1230013-1281741" "chr19-39381822-39438328" "chr17-7832492-7859042" "chr19-12774552-12797715" ...
  ..@ meta.features     :'data.frame':  105913 obs. of  2 variables:
  ..@ misc              : NULL

如fragments矩阵信息

head(pbmc@assays$ATAC@counts)
6 x 10412 sparse Matrix of class "dgCMatrix"
   [[ suppressing 76 column names 'AAACAGCCAAGGAATC-1', 'AAACAGCCAATCCCTT-1', 'AAACAGCCAATGCGCT-1' ... ]]
                                                                                                                                                                    
chr1-10109-10357   . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-180730-181630 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-191491-191736 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-267816-268196 . . . . . . . . . . . . . . . 2 . . . . . . . . . . 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-586028-586373 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-629721-630172 . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
                               
chr1-10109-10357   . . . ......
chr1-180730-181630 . . . ......
chr1-191491-191736 . . . ......
chr1-267816-268196 . . . ......
chr1-586028-586373 . . . ......
chr1-629721-630172 . . . ......

 .....suppressing 10336 columns in show(); maybe adjust 'options(max.print= *, width = *)'
 ..............................

如注释信息:

 head(pbmc@assays$ATAC@annotation)
GRanges object with 6 ranges and 5 metadata columns:
                  seqnames        ranges strand |           tx_id   gene_name         gene_id   gene_biotype     type
                     <Rle>     <IRanges>  <Rle> |     <character> <character>     <character>    <character> <factor>
  ENSE00001489430     chrX 276322-276394      + | ENST00000399012      PLCXD1 ENSG00000182378 protein_coding     exon
  ENSE00001536003     chrX 276324-276394      + | ENST00000484611      PLCXD1 ENSG00000182378 protein_coding     exon
  ENSE00002160563     chrX 276353-276394      + | ENST00000430923      PLCXD1 ENSG00000182378 protein_coding     exon
  ENSE00001750899     chrX 281055-281121      + | ENST00000445062      PLCXD1 ENSG00000182378 protein_coding     exon
  ENSE00001489388     chrX 281192-281684      + | ENST00000381657      PLCXD1 ENSG00000182378 protein_coding     exon
  ENSE00001719251     chrX 281194-281256      + | ENST00000429181      PLCXD1 ENSG00000182378 protein_coding     exon
  -------
  seqinfo: 25 sequences from hg38 genome

理解Seurat的数据是很重要的,不然很多分析数据可能不知道如何提取调用,也不知道函数做了什么。在这之后,我们进行ATAC数据的标准分析。

# ATAC analysis
# We exclude the first dimension as this is typically correlated with sequencing depth
DefaultAssay(pbmc) <- "ATAC"
pbmc <- RunTFIDF(pbmc)
pbmc <- FindTopFeatures(pbmc, min.cutoff = 'q0')
pbmc <- RunSVD(pbmc)
pbmc <- RunUMAP(pbmc, reduction = 'lsi', dims = 2:50, reduction.name = "umap.atac", reduction.key = "atacUMAP_")

注意,这里的以及所有特征选择之后的再次降维用到的维度dims,都需要谨慎选择,我们知道每个坐标值都有一系列特征支持程度(如loading值),可以反映出该维度代表的生物学意义。如这里的dims = 2:50,排除了第一个维度,因为作者发现它通常与排序深度相关

pbmc@reductions$lsi@feature.loadings[1:4,1:4]
                              LSI_1         LSI_2         LSI_3         LSI_4
chr19-1230013-1281741   0.008759956  0.0022949972 -0.0008663089 -0.0019122743
chr19-39381822-39438328 0.008735519 -0.0007909501 -0.0018568479 -0.0018096328
chr17-7832492-7859042   0.008713235  0.0012451020 -0.0006112821  0.0004232284
chr19-12774552-12797715 0.008700547  0.0015833131 -0.0014307690  0.0013240653

用Signac分析过scATAC的同学对这里的流程不会感到陌生,如果是第一次接触ATAC分析会觉得比较陌生了,如RunTFIDF是什么?这时候我们可以?RunTFIDF来查看具体信息以及参考文献。

?RunTFIDF


method
Which TF-IDF implementation to use. Choice of:
*   1: The TF-IDF implementation used by Stuart & Butler et al. 2019 (https://doi.org/10.1101/460147). This computes *\log(TF \times IDF)*.
*   2: The TF-IDF implementation used by Cusanovich & Hill et al. 2018 (https://doi.org/10.1016/j.cell.2018.06.052). This computes *TF \times (\log(IDF))*
*   3: The log-TF method used by Andrew Hill (http://andrewjohnhill.com/blog/2019/05/06/dimensionality-reduction-for-scatac-data/). This computes *\log(TF) \times \log(IDF)*.
*   4: The 10x Genomics method (no TF normalization). This computes *IDF*.

我们计算一个WNN图,表示RNA和ATAC-seq模式的加权组合。我们将此图用于UMAP的可视化和聚类。我们在Seurat 4.0 || 分析scRNA和表面抗体数据
中查看过FindMultiModalNeighbors之后的数据结构。

pbmc <- FindMultiModalNeighbors(pbmc, reduction.list = list("pca", "lsi"), dims.list = list(1:50, 2:50))
Calculating cell-specific modality weights
Finding 20 nearest neighbors for each modality.
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=05s  
Calculating kernel bandwidths
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=01s  
Finding multimodal neighbors
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=20s  
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=01s  
Constructing multimodal KNN graph
Constructing multimodal SNN graph

 pbmc <- RunUMAP(pbmc, nn.name = "weighted.nn", reduction.name = "wnn.umap", reduction.key = "wnnUMAP_")
06:41:32 UMAP embedding parameters a = 0.9922 b = 1.112
06:41:33 Commencing smooth kNN distance calibration using 1 thread
06:41:35 Initializing from normalized Laplacian + noise
06:41:36 Commencing optimization for 200 epochs, with 308698 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
06:41:42 Optimization finished

pbmc <- FindClusters(pbmc, graph.name = "wsnn", algorithm = 3, verbose = FALSE)

这里聚类用的算法是SLM

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.

这些事图聚类的话语体系,我们知道在构建了细胞间的图结构之后,要聚类就需要计算细胞间的相互关系,这里是网略数据科学的一个重要领域:社区发现(community detection),如这里的SLM (smart local moving)算法是一种在大型网络中进行社区发现(或聚类)的算法。SLM算法最大化了所谓的模块化函数。该算法已成功应用于具有数千万个节点和数亿条边的网络。算法的细节记录在一篇论文中(Waltman & Van Eck, 2013)。在igraph中实现SLM可以这样:

#devtools::install_github("chen198328/slm")

library(igraph)
library(slm)
net <- graph_from_adjacency_matrix(pbmc[["wsnn"]]) 
slm.net <-slm.community(net)

当然这对大部分不做算法调整的同学来讲并没有什么用,这里只是说明我们的聚类是在图空间中进行的。

接下来我们对细胞群进行注释。注意,您还可以使用我们构建好的PBMC参考数据集来进行注释,可以用Seurat(vignette见官网)或自动化web工具:Azimuth。

# perform sub-clustering on cluster 6 to find additional structure
pbmc <- FindSubCluster(pbmc, cluster = 6, graph.name = "wsnn", algorithm = 3)
Idents(pbmc) <- "sub.cluster"

指定某群在聚类这个函数好用啊

table(pbmc$seurat_clusters)

   0    1    2    3    4    5    6    7    8    9   10   11   12   13   14   15   16   17   18   19   20   21 
2386 1159  973  606  558  502  498  470  438  438  372  352  329  285  256  198  163  142  137  106   26   18 
> table(pbmc$sub.cluster)

   0    1   10   11   12   13   14   15   16   17   18   19    2   20   21    3    4    5  6_0  6_1    7    8    9 
2386 1159  372  352  329  285  256  198  163  142  137  106  973   26   18  606  558  502  274  224  470  438  438 

其他没变,cluster被分成了两组:6_0 ,6_1

# add annotations
pbmc <- RenameIdents(pbmc, '19' = 'pDC','20' = 'HSPC','15' = 'cDC')
pbmc <- RenameIdents(pbmc, '0' = 'CD14 Mono', '8' ='CD14 Mono', '5' = 'CD16 Mono')
pbmc <- RenameIdents(pbmc, '17' = 'Naive B', '11' = 'Intermediate B', '10' = 'Memory B', '21' = 'Plasma')
pbmc <- RenameIdents(pbmc, '7' = 'NK')
pbmc <- RenameIdents(pbmc, '4' = 'CD4 TCM', '13'= "CD4 TEM", '3' = "CD4 TCM", '16' ="Treg", '1' ="CD4 Naive", '14' = "CD4 Naive")
pbmc <- RenameIdents(pbmc, '2' = 'CD8 Naive', '9'= "CD8 Naive", '12' = 'CD8 TEM_1', '6_0' = 'CD8 TEM_2', '6_1' ='CD8 TEM_2')
pbmc <- RenameIdents(pbmc, '18' = 'MAIT')
pbmc <- RenameIdents(pbmc, '6_2' ='gdT', '6_3' = 'gdT')
pbmc$celltype <- Idents(pbmc)


pbmc
An object of class Seurat 
166710 features across 10412 samples within 3 assays 
Active assay: ATAC (105913 features, 105913 variable features)
 2 other assays present: RNA, SCT
 5 dimensional reductions calculated: pca, umap.rna, lsi, umap.atac, wnn.umap

head(pbmc@meta.data)
                      orig.ident nCount_RNA nFeature_RNA percent.mt nCount_ATAC nFeature_ATAC replicate nCount_SCT nFeature_SCT SCT.weight wsnn_res.0.8
AAACAGCCAAGGAATC-1 SeuratProject       8380         3308   7.470167       55550         13867      para       4685         2693  0.4514450            1
AAACAGCCAATCCCTT-1 SeuratProject       3771         1896  10.527711       20485          7247      para       3781         1895  0.5069799            4
AAACAGCCAATGCGCT-1 SeuratProject       6876         2904   6.457243       16674          6528      para       4686         2769  0.4324867            1
AAACAGCCACCAACCG-1 SeuratProject       5415         2282   6.500462        7658          3323      ctrl       4486         2278  0.4738122            2
AAACAGCCAGGATAAC-1 SeuratProject       2759         1353   6.922798       10355          4267      ctrl       3332         1352  0.5361155            1
AAACAGCCAGTAGGTG-1 SeuratProject       7614         3061   6.895193       39441         11628      ctrl       4711         2764  0.5273038            9
                   seurat_clusters sub.cluster  celltype
AAACAGCCAAGGAATC-1               1           1 CD4 Naive
AAACAGCCAATCCCTT-1               4           4   CD4 TCM
AAACAGCCAATGCGCT-1               1           1 CD4 Naive
AAACAGCCACCAACCG-1               2           2 CD8 Naive
AAACAGCCAGGATAAC-1               1           1 CD4 Naive
AAACAGCCAGTAGGTG-1               9           9 CD8 Naive

基于基因表达可视化聚类,ATAC-seq,或WNN分析。与前面的分析相比,差异更加微妙(您可以探索权重,它比我们的CITE-seq示例中更均匀地分割),但是我们发现WNN提供了最清晰的细胞状态分离。


p1 <- DimPlot(pbmc, reduction = "umap.rna", group.by = "celltype", label = TRUE, label.size = 2.5, repel = TRUE) + ggtitle("RNA")
p2 <- DimPlot(pbmc, reduction = "umap.atac", group.by = "celltype", label = TRUE, label.size = 2.5, repel = TRUE) + ggtitle("ATAC")
p3 <- DimPlot(pbmc, reduction = "wnn.umap", group.by = "celltype", label = TRUE, label.size = 2.5, repel = TRUE) + ggtitle("WNN")
p1 + p2 + p3 & NoLegend() & theme(plot.title = element_text(hjust = 0.5))

例如,ATAC-seq数据有助于CD4和CD8 T细胞状态的分离,这是由于多个基因座的存在。在不同的T细胞亚型之间表现出不同的可及性。例如,我们可以利用Signac可视化工具,将CD8A位点的“伪体(pseudobulk)”轨迹与基因表达水平的小提琴图一起可视化。

## to make the visualization easier, subset T cell clusters
celltype.names <- levels(pbmc)
tcell.names <- grep("CD4|CD8|Treg", celltype.names,value = TRUE)
tcells <- subset(pbmc, idents = tcell.names)
CoveragePlot(tcells, region = 'CD8A', features = 'CD8A', assay = 'ATAC', expression.assay = 'SCT', peaks = FALSE)

接下来,我们将检查每个细胞的可达区域,以确定富集基序(enriched motifs)。正如Signac motifs vignette中所描述的,有几种方法可以做到这一点,但我们将使用Greenleaf实验室的chromVAR包。这将计算已知motifs的每个单元的可访问性得分,并将这些得分作为Seurat对象中的第三个分析(chromVAR)添加。

再走下去,请确保安装了以下软件包。

我先library一下看看哪些是安装过的。

library(chromVAR)
library(JASPAR2020)
library(TFBSTools)
library(motifmatchr)
library(BSgenome.Hsapiens.UCSC.hg38)

针对没安装的,我们可以这样来安装。

remotes::install_github("immunogenomics/presto")
BiocManager::install(c("chromVAR", "TFBSTools", "JASPAR2020", "motifmatchr", "BSgenome.Hsapiens.UCSC.hg38"))  
# Scan the DNA sequence of each peak for the presence of each motif, and create a Motif object
DefaultAssay(pbmc) <- "ATAC"
pwm_set <- getMatrixSet(x = JASPAR2020, opts = list(species = 9606, all_versions = FALSE))
motif.matrix <- CreateMotifMatrix(features = granges(pbmc), pwm = pwm_set, genome = 'hg38', use.counts = FALSE)
motif.object <- CreateMotifObject(data = motif.matrix, pwm = pwm_set)
pbmc <- SetAssayData(pbmc, assay = 'ATAC', slot = 'motifs', new.data = motif.object)

# Note that this step can take 30-60 minutes 
pbmc <- RunChromVAR(
  object = pbmc,
  genome = BSgenome.Hsapiens.UCSC.hg38
)

 pbmc
An object of class Seurat 
166710 features across 10412 samples within 3 assays 
Active assay: ATAC (105913 features, 105913 variable features)
 2 other assays present: RNA, SCT
 5 dimensional reductions calculated: pca, umap.rna, lsi, umap.atac, wnn.umap

最后,我们探索多模态数据集来识别每个细胞状态的关键调节器(key regulators)。配对数据提供了一个独特的机会来识别满足多种标准的转录因子(TFs),帮助缩小假定的调控因子的名单列表。我们的目标是在RNA测量中识别在多种细胞类型中表达丰富的TFs,同时在ATAC测量中富集其motifs 的可达性。

作为阳性对照,CCAAT增强子结合蛋白(CEBP)家族蛋白,包括TF CEBPB,已多次证明在单核细胞和树突状细胞等髓系细胞的分化和功能中发挥重要作用。我们可以看到CEBPB的表达和编码CEBPB结合位点的MA0466.2.4基序的可及性都在单核细胞中富集。

#returns MA0466.2
motif.name <- ConvertMotifID(pbmc, name = 'CEBPB')
gene_plot <- FeaturePlot(pbmc, features = "sct_CEBPB", reduction = 'wnn.umap')
motif_plot <- FeaturePlot(pbmc, features = motif.name, min.cutoff = 0, cols = c("lightgrey", "darkred"), reduction = 'wnn.umap')
gene_plot | motif_plot

我们想要量化这个关系,并搜索所有的细胞类型来找到类似的例子。为此,我们将使用presto包来执行快差异分析。我们进行了两项测试 :一项使用基因表达数据,另一项使用chromVAR motif可达性。presto根据Wilcox秩和检验(这也是Seurat的默认方法)计算一个p值,我们将搜索限制为在两个测试中都返回显著结果的TFs。

presto还计算了“AUC”统计值,它反映了每个基因(或基序)作为细胞类型标记的能力。曲线下面积(AUC)的最大值为1表示标记良好。由于基因和基序的AUC统计值是相同的,因此我们将两种测试的AUC值取平均值,并以此来对每种细胞类型的TFs进行排序:

markers_rna <- presto:::wilcoxauc.Seurat(X = pbmc, group_by = 'celltype', assay = 'data', seurat_assay = 'SCT')
markers_motifs <- presto:::wilcoxauc.Seurat(X = pbmc, group_by = 'celltype', assay = 'data', seurat_assay = 'chromvar')
motif.names <- markers_motifs$feature
colnames(markers_rna) <- paste0("RNA.", colnames(markers_rna))
colnames(markers_motifs) <- paste0("motif.", colnames(markers_motifs))
markers_rna$gene <- markers_rna$RNA.feature
markers_motifs$gene <- ConvertMotifID(pbmc, id = motif.names)
head(markers_motifs)
  motif.feature motif.group motif.avgExpr motif.logFC motif.statistic motif.auc   motif.pval   motif.padj motif.pct_in
1      MA0030.1   CD14 Mono    -0.2070450  -0.2837042         8748968 0.4082863 4.331768e-47 5.397656e-47          100
2      MA0031.1   CD14 Mono    -0.6256700  -0.8643914         5368273 0.2505201 0.000000e+00 0.000000e+00          100
3      MA0051.1   CD14 Mono     0.1237673   0.1974597        11955125 0.5579074 9.048795e-20 1.045235e-19          100
4      MA0057.1   CD14 Mono     0.7245745   1.0173693        16968911 0.7918847 0.000000e+00 0.000000e+00          100
5      MA0059.1   CD14 Mono     0.9826472   1.3781270        17525266 0.8178480 0.000000e+00 0.000000e+00          100
6      MA0066.1   CD14 Mono     1.6772753   2.3573633        19347910 0.9029049 0.000000e+00 0.000000e+00          100
  motif.pct_out        gene
1           100       FOXF2
2           100       FOXD1
3           100        IRF2
4           100 MZF1(var.2)
5           100    MAX::MYC
6           100       PPARG

寻找toptf的函数

# a simple function to implement the procedure above
topTFs <- function(celltype, padj.cutoff = 1e-2) {
  ctmarkers_rna <- dplyr::filter(
    markers_rna, RNA.group == celltype, RNA.padj < padj.cutoff, RNA.logFC > 0) %>% 
    arrange(-RNA.auc)
  ctmarkers_motif <- dplyr::filter(
    markers_motifs, motif.group == celltype, motif.padj < padj.cutoff, motif.logFC > 0) %>% 
    arrange(-motif.auc)
  top_tfs <- inner_join(
    x = ctmarkers_rna[, c(2, 11, 6, 7)], 
    y = ctmarkers_motif[, c(2, 1, 11, 6, 7)], by = "gene"
  )
  top_tfs$avg_auc <- (top_tfs$RNA.auc + top_tfs$motif.auc) / 2
  top_tfs <- arrange(top_tfs, -avg_auc)
  return(top_tfs)
}

我们现在可以计算和可视化任何细胞类型的putative regulators。我们恢复了成熟的调控因子,包括用于NK细胞的TBX21(这里的背景知识很重要啊),浆细胞的IRF4,造血祖细胞的SOX4,B细胞的EBF1和PAX5,pDC的IRF8和TCF4。我们相信,类似的策略可以用来帮助关注不同系统中一组putative regulators。


# identify top markers in NK and visualize
head(topTFs("NK"), 3)

head(topTFs("NK"), 3)
  RNA.group  gene   RNA.auc      RNA.pval motif.group motif.feature motif.auc    motif.pval   avg_auc
1        NK TBX21 0.7269867  0.000000e+00          NK      MA0690.1 0.9175550 3.489416e-206 0.8222709
2        NK EOMES 0.5898072 8.133395e-101          NK      MA0800.1 0.9238892 2.013461e-212 0.7568482
3        NK RUNX3 0.7712620 9.023079e-121          NK      MA0684.2 0.6923503  3.069778e-45 0.7318062
motif.name <- ConvertMotifID(pbmc, name = 'TBX21')
gene_plot <- FeaturePlot(pbmc, features = "sct_TBX21", reduction = 'wnn.umap')
motif_plot <- FeaturePlot(pbmc, features = motif.name, min.cutoff = 0, cols = c("lightgrey", "darkred"), reduction = 'wnn.umap')
gene_plot | motif_plot
# identify top markers in pDC and visualize
head(topTFs("pDC"), 3)

 head(topTFs("pDC"), 3)
  RNA.group  gene   RNA.auc      RNA.pval motif.group motif.feature motif.auc   motif.pval   avg_auc
1       pDC  TCF4 0.9998833 3.347777e-163         pDC      MA0830.2 0.9952958 3.912770e-69 0.9975896
2       pDC  IRF8 0.9905372 2.063258e-124         pDC      MA0652.1 0.8714643 1.143055e-39 0.9310008
3       pDC RUNX2 0.9754498 7.815119e-112         pDC      MA0511.2 0.8355034 1.126551e-32 0.9054766
motif.name <- ConvertMotifID(pbmc, name = 'TCF4')
gene_plot <- FeaturePlot(pbmc, features = "sct_TCF4", reduction = 'wnn.umap')
motif_plot <- FeaturePlot(pbmc, features = motif.name, min.cutoff = 0, cols = c("lightgrey", "darkred"), reduction = 'wnn.umap')
gene_plot | motif_plot
# identify top markers in HSPC and visualize
head(topTFs("CD16 Mono"),3)

RNA.group  gene   RNA.auc      RNA.pval motif.group motif.feature motif.auc    motif.pval   avg_auc
1 CD16 Mono TBX21 0.6550599 8.750032e-193   CD16 Mono      MA0690.1 0.8896766 2.483767e-191 0.7723682
2 CD16 Mono EOMES 0.5862998  3.802279e-99   CD16 Mono      MA0800.1 0.8922904 7.043330e-194 0.7392951
3 CD16 Mono RUNX3 0.7182799  7.394573e-84   CD16 Mono      MA0684.2 0.6753424  3.175696e-40 0.6968111
motif.name <- ConvertMotifID(pbmc, name = 'SPI1')
gene_plot <- FeaturePlot(pbmc, features = "sct_SPI1", reduction = 'wnn.umap')
motif_plot <- FeaturePlot(pbmc, features = motif.name, min.cutoff = 0, cols = c("lightgrey", "darkred"), reduction = 'wnn.umap')
gene_plot | motif_plot
> # identify top markers in other cell types
> head(topTFs("Naive B"), 3)
  RNA.group   gene   RNA.auc     RNA.pval motif.group motif.feature motif.auc   motif.pval   avg_auc
1   Naive B   EBF1 0.9604105 0.000000e+00     Naive B      MA0154.4 0.7562941 8.038044e-26 0.8583523
2   Naive B POU2F2 0.6338272 2.833732e-09     Naive B      MA0507.1 0.9702895 7.975628e-83 0.8020583
3   Naive B   TCF4 0.7139073 2.689186e-41     Naive B      MA0830.2 0.8641510 2.153784e-50 0.7890291
> head(topTFs("HSPC"), 3)
  RNA.group  gene   RNA.auc     RNA.pval motif.group motif.feature motif.auc   motif.pval   avg_auc
1      HSPC  SOX4 0.9869221 7.766427e-71        HSPC      MA0867.2 0.6967552 5.188151e-04 0.8418387
2      HSPC GATA2 0.7884615 0.000000e+00        HSPC      MA0036.3 0.8240827 1.084319e-08 0.8062721
3      HSPC MEIS1 0.8830582 0.000000e+00        HSPC      MA0498.2 0.7048912 3.010760e-04 0.7939747
> head(topTFs("Plasma"), 3)
  RNA.group  gene   RNA.auc     RNA.pval motif.group motif.feature motif.auc   motif.pval   avg_auc
1    Plasma  IRF4 0.8189901 5.206829e-35      Plasma      MA1419.1 0.9821051 1.452382e-12 0.9005476
2    Plasma MEF2C 0.9070644 4.738760e-12      Plasma      MA0497.1 0.7525389 2.088028e-04 0.8298016
3    Plasma  TCF4 0.8052937 5.956053e-12      Plasma      MA0830.2 0.7694984 7.585013e-05 0.7873960

https://satijalab.org/seurat/v4.0/weighted_nearest_neighbor_analysis.html
https://monkeylearn.com/blog/what-is-tf-idf/
https://www.biorxiv.org/content/10.1101/460147v1.full
http://www.ludowaltman.nl/slm/#:~:text=The%20SLM%20algorithm%20has%20been%20implemented%20in%20the,be%20run%20on%20any%20system%20with%20Java%20support.
https://www.cwts.nl/blog?article=n-r2u2a4
https://github.com/chen198328/slm

上一篇下一篇

猜你喜欢

热点阅读