上一篇文章里面简单学习了一下表达矩阵的提取，顺便探索了一下SummarizedExperiment对象。
今天学习下用TCGAbiolinks做差异分析。
加载R包和数据
rm(list = ls())
library(SummarizedExperiment)
## Loading required package: MatrixGenerics
## Loading required package: matrixStats
##
## Attaching package: 'MatrixGenerics'
## The following objects are masked from 'package:matrixStats':
##
## colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse,
## colCounts, colCummaxs, colCummins, colCumprods, colCumsums,
## colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs,
## colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats,
## colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds,
## colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads,
## colWeightedMeans, colWeightedMedians, colWeightedSds,
## colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet,
## rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods,
## rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps,
## rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins,
## rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks,
## rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars,
## rowWeightedMads, rowWeightedMeans, rowWeightedMedians,
## rowWeightedSds, rowWeightedVars
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library(TCGAbiolinks)
首先是查询、下载、整理，但是这一步我们在之前已经做好了，直接加载就可以了！
下载方法：请翻看之前的推文
# 查询
query <- GDCquery(project = "TCGA-COAD",
data.category = "Transcriptome Profiling",
data.type = "Gene Expression Quantification",
workflow.type = "STAR - Counts"
)
# 下载
GDCdownload(query, files.per.chunk = 100) #每次下载100个文件
# 整理
GDCprepare(query,save = T,save.filename = "TCGA-COAD_mRNA.Rdata")
我们已经下载好了，就直接加载即可：
load("TCGA-mRNA/TCGA-COAD_mRNA.Rdata")
通常我们会区分mRNA和lncRNA，所以我们这里只选择mRNA即可，方法在上一篇也说过了，非常简单！直接对SummarizedExperiment对象取子集即可！
se_mrna <- data[rowData(data)$gene_type == "protein_coding",]
se_mrna
## class: RangedSummarizedExperiment
## dim: 19962 521
## metadata(1): data_release
## assays(6): unstranded stranded_first ... fpkm_unstrand fpkm_uq_unstrand
## rownames(19962): ENSG00000000003.15 ENSG00000000005.6 ...
## ENSG00000288674.1 ENSG00000288675.1
## rowData names(10): source type ... hgnc_id havana_gene
## colnames(521): TCGA-A6-5664-01A-21R-1839-07
## TCGA-D5-6530-01A-11R-1723-07 ... TCGA-A6-2683-01A-01R-0821-07
## TCGA-A6-2683-11A-01R-A32Z-07
## colData names(107): barcode patient ... paper_vascular_invasion_present
## paper_vital_status
数据预处理
在进行差异分析前进行一些预处理。
dim(se_mrna)
## [1] 19962 521
首先根据spearman相关系数去除异常值：
coad_coroutliers <- TCGAanalyze_Preprocessing(se_mrna,cor.cut = 0.7)
## Number of outliers: 0
dim(coad_coroutliers)
## [1] 19962 521
还会生成一张相关图：Array Array Intensity correlation (AAIC)：
image.png
接下来进行标准化：
# normalization of genes
coadNorm <- TCGAanalyze_Normalization(
tabDF = coad_coroutliers,
geneInfo = geneInfoHT
)
## I Need about 127 seconds for this Complete Normalization Upper Quantile [Processing 80k elements /s]
## Step 1 of 4: newSeqExpressionSet ...
## Step 2 of 4: withinLaneNormalization ...
## Step 3 of 4: betweenLaneNormalization ...
## Step 4 of 4: exprs ...
会使用EDASeq包中的方法：
EDASeq::newSeqExpressionSet
EDASeq::withinLaneNormalization
EDASeq::betweenLaneNormalization
EDASeq::counts
dim(coadNorm)
## [1] 19469 521
然后过滤掉低表达的基因：
# quantile filter of genes
coadFilt <- TCGAanalyze_Filtering(
tabDF = coadNorm,
method = "quantile",
qnt.cut = 0.25
)
看看一通操作下来后还剩多少基因？
dim(coadFilt)
## [1] 14600 521
从最开始的19962变成了现在的14600，过滤掉了5000+基因......
最后是根据肿瘤组织和正常组织进行分组：
这里我们只选择了实体瘤和部分正常组织。如果你想选择更多，只要在typesample参数中添加更多类型即可。
可选类型见下图，也是根据TCGA-barcode进行判断的：
image.png
# selection of normal samples "NT"
samplesNT <- TCGAquery_SampleTypes(
barcode = colnames(coadFilt),
typesample = c("NT")
)
# selection of tumor samples "TP"
samplesTP <- TCGAquery_SampleTypes(
barcode = colnames(coadFilt),
typesample = c("TP")
)
所以分组这一步我们还是自己搞定！就是根据barcode的第14,15位数，结合上面那张图判断，毫无难度。
image.png
# 小于10就是tumor
samplesTumor <- as.numeric(substr(colnames(coadFilt),14,15))<10
差异分析
非常简单，支持edgeR和limma两种方法，当然也可以无缝连接DESeq2进行差异分析！
# Diff.expr.analysis (DEA)
coadDEGs <- TCGAanalyze_DEA(
mat1 = coadFilt[,!samplesTumor], # normal矩阵
mat2 = coadFilt[,samplesTumor], # tumor矩阵
Cond1type = "Normal",
Cond2type = "Tumor",
fdr.cut = 0.01,
logFC.cut = 1,
pipeline = "edgeR", # limma
method = "glmLRT"
)
## Batch correction skipped since no factors provided
## ----------------------- DEA -------------------------------
## o 41 samples in Cond1type Normal
## o 480 samples in Cond2type Tumor
## o 14600 features as miRNA or genes
## This may take some minutes...
## ----------------------- END DEA -------------------------------
差异分析就做好了！结果非常完美，同时提供了gene_name和gene_type，也就是说我们一开始不取子集也是可以的~~，最后再取也行！
image.png
使用DESeq2进行差异分析
连接DESeq2那真是太简单了，无缝衔接！！
library(DESeq2)
直接把SummarizedExperiment对象传给DESeqDataSet()函数即可。
不过需要分组信息，这个需要我们手动制作一下。
制作分组信息
其实我们的对象中包含了sample_type这一列信息，就在coldata中，但是有点过于详细了。
table(colData(se_mrna)$sample_type)
##
## Metastatic Primary Tumor Recurrent Tumor Solid Tissue Normal
## 1 478 1 41
我们给它修改一下~
new_type <- ifelse(colData(se_mrna)$sample_type == "Solid Tissue Normal",
"Normal",
"Tumor")
colData(se_mrna)$sample_type <- new_type
table(colData(se_mrna)$sample_type)
##
## Normal Tumor
## 41 480
这样就把分组搞定了！skr!
差异分析
然后就是愉快的进行差异分析~
ddsSE <- DESeqDataSet(se_mrna, design = ~ sample_type)
## renaming the first element in assays to 'counts'
ddsSE
## class: DESeqDataSet
## dim: 19962 521
## metadata(2): data_release version
## assays(6): counts stranded_first ... fpkm_unstrand fpkm_uq_unstrand
## rownames(19962): ENSG00000000003.15 ENSG00000000005.6 ...
## ENSG00000288674.1 ENSG00000288675.1
## rowData names(10): source type ... hgnc_id havana_gene
## colnames(521): TCGA-A6-5664-01A-21R-1839-07
## TCGA-D5-6530-01A-11R-1723-07 ... TCGA-A6-2683-01A-01R-0821-07
## TCGA-A6-2683-11A-01R-A32Z-07
## colData names(107): barcode patient ... paper_vascular_invasion_present
## paper_vital_status
先过滤，这里我们简单点，
keep <- rowSums(counts(ddsSE)) >= 50
ddsSE <- ddsSE[keep,]
ddsSE
## class: DESeqDataSet
## dim: 18820 521
## metadata(2): data_release version
## assays(6): counts stranded_first ... fpkm_unstrand fpkm_uq_unstrand
## rownames(18820): ENSG00000000003.15 ENSG00000000005.6 ...
## ENSG00000288674.1 ENSG00000288675.1
## rowData names(10): source type ... hgnc_id havana_gene
## colnames(521): TCGA-A6-5664-01A-21R-1839-07
## TCGA-D5-6530-01A-11R-1723-07 ... TCGA-A6-2683-01A-01R-0821-07
## TCGA-A6-2683-11A-01R-A32Z-07
## colData names(107): barcode patient ... paper_vascular_invasion_present
## paper_vital_status
确定谁和谁比，我们设定Tumor-Normal。
ddsSE$sample_type <- factor(ddsSE$sample_type, levels = c("Tumor","Normal"))
接下来就可以进行差异分析了：
dds <- DESeq(ddsSE)
## estimating size factors
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## final dispersion estimates
## fitting model and testing
## -- replacing outliers and refitting for 1544 genes
## -- DESeq argument 'minReplicatesForReplace' = 7
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
res <- results(dds, contrast = c("sample_type","Tumor","Normal"))
res
## log2 fold change (MLE): sample_type Tumor vs Normal
## Wald test p-value: sample_type Tumor vs Normal
## DataFrame with 18820 rows and 6 columns
## baseMean log2FoldChange lfcSE stat pvalue
##
## ENSG00000000003.15 5198.688 0.456284 0.1445636 3.15628 1.59793e-03
## ENSG00000000005.6 42.064 0.414580 0.3014120 1.37546 1.68989e-01
## ENSG00000000419.13 1743.704 0.849473 0.1134251 7.48929 6.92491e-14
## ENSG00000000457.14 463.909 -0.261646 0.0690276 -3.79046 1.50371e-04
## ENSG00000000460.17 328.546 1.272811 0.0940574 13.53229 1.00837e-41
## ... ... ... ... ... ...
## ENSG00000288658.1 5.317261 -1.45986802 0.274148 -5.3251160 1.00889e-07
## ENSG00000288660.1 4.648268 1.47152585 0.450554 3.2660389 1.09063e-03
## ENSG00000288669.1 0.143343 -0.13840184 1.339247 -0.1033431 9.17691e-01
## ENSG00000288674.1 3.201667 0.00548347 0.174731 0.0313824 9.74965e-01
## ENSG00000288675.1 15.048025 2.01434132 0.174631 11.5348224 8.80688e-31
## padj
##
## ENSG00000000003.15 2.75622e-03
## ENSG00000000005.6 2.13290e-01
## ENSG00000000419.13 3.14116e-13
## ENSG00000000457.14 2.95220e-04
## ENSG00000000460.17 2.77449e-40
## ... ...
## ENSG00000288658.1 2.74461e-07
## ENSG00000288660.1 1.92231e-03
## ENSG00000288669.1 9.29645e-01
## ENSG00000288674.1 9.78866e-01
## ENSG00000288675.1 1.20917e-29
结果很棒，不过没有gene_symbol了，需要自己添加哦~