生信技能树R作业答案-高级

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1 安装一些R包

数据包： ALL, CLL, pasilla, airway 软件包：limma，DESeq2，clusterProfiler 工具包：reshape2 绘图包：ggplot2

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rm(list = ls())
if(F){
  source("http://bioconductor.org/biocLite.R")
  options("repos" = c(CRAN="https://mirrors.tuna.tsinghua.edu.cn/CRAN/"))
  options(BioC_mirror="http://mirrors.ustc.edu.cn/bioc/")#修改镜像，安装会加速
  BiocManager::install("clusterProfiler")
  BiocManager::install("ComplexHeatmap")
  BiocManager::install("maftools")
  BiocManager::install("reshape2")
}

2了解ExpressionSet对象.

比如CLL包里面就有data(sCLLex) 找到它包含的元素，提取其表达矩阵(使用exprs函数)，查看其大小

A.参考：http://www.bio-info-trainee.com/bioconductor_China/software/limma.html B.参考：https://github.com/bioconductor-china/basic/blob/master/ExpressionSet.md

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library(CLL)
data("sCLLex")
View(sCLLex)#查看其包含的元素
sCLLex#查看包含的元素
## ExpressionSet (storageMode: lockedEnvironment)
## assayData: 12625 features, 22 samples 
##   element names: exprs 
## protocolData: none
## phenoData
##   sampleNames: CLL11.CEL CLL12.CEL ... CLL9.CEL (22 total)
##   varLabels: SampleID Disease
##   varMetadata: labelDescription
## featureData: none
## experimentData: use 'experimentData(object)'
## Annotation: hgu95av2

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exp <- exprs(sCLLex)#提取表达矩阵
dim(exp)#查看其大小
## [1] 12625    22

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group <- pData(sCLLex)#提取分组信息
table(group)#设计矩阵
##         Disease
## SampleID progres. stable
##    CLL11        1      0
##    CLL12        0      1
##    CLL13        1      0
##    CLL14        1      0
##    CLL15        1      0
##    CLL16        1      0
##    CLL17        0      1
##    CLL18        0      1
##    CLL19        1      0
##    CLL2         0      1
##    CLL20        0      1
##    CLL21        1      0
##    CLL22        0      1
##    CLL23        1      0
##    CLL24        0      1
##    CLL3         1      0
##    CLL4         1      0
##    CLL5         1      0
##    CLL6         1      0
##    CLL7         1      0
##    CLL8         1      0
##    CLL9         0      1

3了解 str,head,help函数

作用于 第二步提取到的表达矩阵

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str(exp)
##  num [1:12625, 1:22] 5.74 2.29 3.31 1.09 7.54 ...
##  - attr(*, "dimnames")=List of 2
##   ..$ : chr [1:12625] "1000_at" "1001_at" "1002_f_at" "1003_s_at" ...
##   ..$ : chr [1:22] "CLL11.CEL" "CLL12.CEL" "CLL13.CEL" "CLL14.CEL" ...

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head(exp)
##           CLL11.CEL CLL12.CEL CLL13.CEL CLL14.CEL CLL15.CEL CLL16.CEL
## 1000_at    5.743132  6.219412  5.523328  5.340477  5.229904  4.920686
## 1001_at    2.285143  2.291229  2.287986  2.295313  2.662170  2.278040
## 1002_f_at  3.309294  3.318466  3.354423  3.327130  3.365113  3.568353
## 1003_s_at  1.085264  1.117288  1.084010  1.103217  1.074243  1.073097
## 1004_at    7.544884  7.671801  7.474025  7.152482  6.902932  7.368660
## 1005_at    5.083793  7.610593  7.631311  6.518594  5.059087  4.855161
##           CLL17.CEL CLL18.CEL CLL19.CEL CLL20.CEL CLL21.CEL CLL22.CEL
## 1000_at    5.325348  4.826131  5.212387  5.285830  5.581859  6.251678
## 1001_at    2.350796  2.325163  2.432635  2.256547  2.348389  2.263849
## 1002_f_at  3.502440  3.394410  3.617099  3.279726  3.391734  3.306811
## 1003_s_at  1.091264  1.076470  1.132558  1.096870  1.138386  1.061184
## 1004_at    6.456285  6.824862  7.304803  8.757756  6.695295  7.372185
## 1005_at    5.176975  4.874563  4.097580  9.250585  7.657463  7.683677
##           CLL23.CEL CLL24.CEL CLL2.CEL CLL3.CEL CLL4.CEL CLL5.CEL CLL6.CEL
## 1000_at    5.480752  5.216033 5.966942 5.397508 5.281720 5.414718 5.460626
## 1001_at    2.264434  2.344079 2.350073 2.406846 2.341961 2.372928 2.356978
## 1002_f_at  3.341444  3.798335 3.427736 3.453564 3.412944 3.411922 3.396466
## 1003_s_at  1.046188  1.082169 1.501367 1.191339 1.139510 1.153548 1.135671
## 1004_at    7.616749  6.839187 7.545673 8.802729 7.307752 7.491507 8.063202
## 1005_at    8.700667  5.546996 9.611601 5.732182 6.483191 6.072558 9.919125
##           CLL7.CEL CLL8.CEL CLL9.CEL
## 1000_at   5.897821 5.253883 5.214155
## 1001_at   2.222276 2.254772 2.358544
## 1002_f_at 3.247276 3.255148 3.365746
## 1003_s_at 1.074631 1.166247 1.151184
## 1004_at   7.014543 8.019108 7.432331
## 1005_at   5.149411 4.989604 5.339848

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help(exp)
## starting httpd help server ... done

4安装并了解 hgu95av2.db 包.

看看 ls(“package:hgu95av2.db”) 后 显示的那些变量

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library(hgu95av2.db)
ls("package:hgu95av2.db")
##  [1] "hgu95av2"              "hgu95av2.db"          
##  [3] "hgu95av2_dbconn"       "hgu95av2_dbfile"      
##  [5] "hgu95av2_dbInfo"       "hgu95av2_dbschema"    
##  [7] "hgu95av2ACCNUM"        "hgu95av2ALIAS2PROBE"  
##  [9] "hgu95av2CHR"           "hgu95av2CHRLENGTHS"   
## [11] "hgu95av2CHRLOC"        "hgu95av2CHRLOCEND"    
## [13] "hgu95av2ENSEMBL"       "hgu95av2ENSEMBL2PROBE"
## [15] "hgu95av2ENTREZID"      "hgu95av2ENZYME"       
## [17] "hgu95av2ENZYME2PROBE"  "hgu95av2GENENAME"     
## [19] "hgu95av2GO"            "hgu95av2GO2ALLPROBES" 
## [21] "hgu95av2GO2PROBE"      "hgu95av2MAP"          
## [23] "hgu95av2MAPCOUNTS"     "hgu95av2OMIM"         
## [25] "hgu95av2ORGANISM"      "hgu95av2ORGPKG"       
## [27] "hgu95av2PATH"          "hgu95av2PATH2PROBE"   
## [29] "hgu95av2PFAM"          "hgu95av2PMID"         
## [31] "hgu95av2PMID2PROBE"    "hgu95av2PROSITE"      
## [33] "hgu95av2REFSEQ"        "hgu95av2SYMBOL"       
## [35] "hgu95av2UNIGENE"       "hgu95av2UNIPROT"

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ls(hgu95av2.db)
## [1] "conn"        "packageName"

5利用注释包找到某基因对应的探针

理解 head(toTable(hgu95av2SYMBOL)) 的用法，找到 TP53 基因对应的探针ID

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head(toTable(hgu95av2SYMBOL))
##    probe_id  symbol
## 1   1000_at   MAPK3
## 2   1001_at    TIE1
## 3 1002_f_at CYP2C19
## 4 1003_s_at   CXCR5
## 5   1004_at   CXCR5
## 6   1005_at   DUSP1

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ids <- toTable(hgu95av2SYMBOL)

5.1 subset函数

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a1 <- subset(ids, ids$symbol=='TP53')

5.2 grep函数

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a4 <- ids[grep("^TP53$", ids$symbol),]

5.3 filter函数(依赖dplyr包)

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a5 <- filter(ids, ids$symbol=='TP53')

6理解基于与探针的对应关系

总共多少个基因，基因最多对应多少个探针， 是哪些基因，是不是因为这些基因很长，所以在其上面设计多个探针呢？总共多少gene

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length(unique(ids$symbol))
## [1] 8585

对应探针多的是那些gene

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ids_fre <- data.frame(table(ids$symbol))
tail(ids_fre[order(ids_fre$Freq),])
##        Var1 Freq
## 8365 YME1L1    7
## 2809  GAPDH    8
## 3638 INPP4A    8
## 4720    MYB    8
## 6010 PTGER3    8
## 7339  STAT1    8

或者用sort命令

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tail(sort(table(ids$symbol)))
## 
## YME1L1  GAPDH INPP4A    MYB PTGER3  STAT1 
##      7      8      8      8      8      8

不管是Agilent芯片，还是Affymetrix芯片，上面设计的探针都非常短。 最长的如Agilent芯片上的探针，往往都是60bp，但是往往一个基因的长 度都好几Kb。因此一般多个探针对应一个基因，取最大表达值探针来作为 基因的表达量。 # 7.找到芯片有而hgu95av2.db中没有对应基因名的探针 第二步提取到的表达矩阵是12625个探针在22个样本的表达量矩阵， 找到那些不在 hgu95av2.db 包收录的对应着SYMBOL的探针。 提示：有1165个探针是没有对应基因名字的。 ## 第一种方法：match函数

7找到芯片有而注释包没有symbole的探针

第二步提取到的表达矩阵是12625个探针在22个样本的表达量矩阵，找到那些不在 hgu95av2.db 包收录的对应着SYMBOL的探针

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dim(exp)
## [1] 12625    22

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dim(hgu95av2SYMBOL)
## [1] 11460     2

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b1 <- exp[-match(ids$probe_id, rownames(exp)),]
dim(b1)
## [1] 1165   22

第二种方法:%in%函数

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b2 <- exp[!(rownames(exp) %in% ids$probe_id),]
b1==b2

8删除注释包没有symbol的探针

删除注释包中没有对应基因名的探针

过滤表达矩阵，删除那1165个没有对应基因名字的探针。

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exp_filter_no_symbol <- as.data.frame(exp[match(ids$probe_id, rownames(exp)),])
exp_filter_no_symbol2 <- as.data.frame(exp[rownames(exp) %in% ids$probe_id,])

9多个探针保留一个

多个探针对应一个基因的情况下，只保留在所有样本里面平均表达量最大的那个探针。
提示，理解 tapply,by,aggregate,split 函数 , 首先对每个基因找到最大表达量的探针。 然后根据得到探针去过滤原始表达矩阵 这种方法能够一定程度上增加差异基因的数目，但也容易造成假阳性的结果 https://blog.csdn.net/tommyhechina/article/details/80468361 合并探针，先看有无NA值，若有，可以删除或填充 先检查有无，若返回值>0,说明有NA值。

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length(which(is.na(exp_filter_no_symbol)))
## [1] 0

结果是0，说明没有NA值。如果有则用impute包来进行填充 exp_symbol<- impute.knn(exp_symbol)$data 下面开始进行合并，用aggregate命令

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ids <- toTable(hgu95av2SYMBOL)
ids$median <- apply(exp_filter_no_symbol, 1, median)
ids <- ids[order(ids$symbol, ids$median, decreasing = T),]
ids_filtered <- ids[!duplicated(ids$symbol),]
dim(ids_filtered)
## [1] 8585    3

10过滤后的表达矩阵行名改为SYMBOL

把过滤后的表达矩阵更改行名为基因的symbol，因为这个时候探针和基因是一对一关系了。

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exp_filter_no_symbol$probe_id <- rownames(exp_filter_no_symbol)
exp_ids <- merge(ids_filtered, exp_filter_no_symbol, by ='probe_id' )
rownames(exp_ids) <- exp_ids$symbol
exp_sym <- exp_ids[,-c(1:3)]

11画第一个和所有样本的基于表达量的图

对第10步得到的表达矩阵进行探索，先画第一个样本的所有基因的表达量的boxplot,hist,density ， 然后画所有样本的 这些图 参考：http://bio-info-trainee.com/tmp/basic_visualization_for_expression_matrix.html 理解ggplot2的绘图语法，数据和图形元素的映射关系

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boxplot(exp_sym$CLL11.CEL)

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boxplot(exp_sym)

用ggplot2来画 ggplot2画图要改变数据形式

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library(reshape2)
exp_g <- melt(exp_ids[,-c(1,3)], id.vars = 'symbol')
exp_g$group <- rep(group$Disease, each = nrow(exp_sym))
colnames(exp_g)[2] <- 'sample'
head(exp_g)
##    symbol    sample    value    group
## 1   MAPK3 CLL11.CEL 5.743132 progres.
## 2    TIE1 CLL11.CEL 2.285143 progres.
## 3 CYP2C19 CLL11.CEL 3.309294 progres.
## 4   CXCR5 CLL11.CEL 7.544884 progres.
## 5   DUSP1 CLL11.CEL 5.083793 progres.
## 6   MMP10 CLL11.CEL 3.252213 progres.

第一个样本的

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library(ggplot2)
## Registered S3 methods overwritten by 'ggplot2':
##   method         from 
##   [.quosures     rlang
##   c.quosures     rlang
##   print.quosures rlang

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sample_1 <- subset(exp_g, exp_g$sample=='CLL2.CEL')
head(sample_1)
##         symbol   sample    value  group
## 120191   MAPK3 CLL2.CEL 5.966942 stable
## 120192    TIE1 CLL2.CEL 2.350073 stable
## 120193 CYP2C19 CLL2.CEL 3.427736 stable
## 120194   CXCR5 CLL2.CEL 7.545673 stable
## 120195   DUSP1 CLL2.CEL 9.611601 stable
## 120196   MMP10 CLL2.CEL 3.380044 stable

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p_s1 <- ggplot(sample_1, aes(x = sample, y = value, fill = 'lightblue'))
p_s1 + geom_boxplot()

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p_s1 + geom_violin()+geom_boxplot(fill = 'white')

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ggplot(sample_1, aes(x = value, fill = 'lightblue'))+
  geom_histogram(bins = 500)

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ggplot(sample_1, aes(x = value, fill = 'lightblue'))+
  geom_density()

下面是所有样本的

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p <- ggplot(exp_g, aes(x = sample, y = value, fill = group))
p + geom_boxplot()

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p + geom_violin()

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ggplot(exp_g, aes(x=value,fill = group))+ geom_histogram(bins = 500)+
       facet_wrap(~group, nrow = 5)

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ggplot(exp_g, aes(x=value,fill = group))+ geom_density()+
  facet_wrap(~group, nrow = 5)

# 12 理解统计学指标mean,median,max,min,sd,var,mad 并计算出每个基因在所有样本的这些统计学指标，最后按照mad值排序，取top 50 mad值的基因，得到列表

12统计学指标

理解统计学指标mean,median,max,min,sd,var,mad并计算出每个基因在所有样本的这些统计学指标，最后按照mad值排序，取top 50 mad值的基因，得到列表

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head(exp_sym)
##         CLL11.CEL CLL12.CEL CLL13.CEL CLL14.CEL CLL15.CEL CLL16.CEL
## MAPK3    5.743132  6.219412  5.523328  5.340477  5.229904  4.920686
## TIE1     2.285143  2.291229  2.287986  2.295313  2.662170  2.278040
## CYP2C19  3.309294  3.318466  3.354423  3.327130  3.365113  3.568353
## CXCR5    7.544884  7.671801  7.474025  7.152482  6.902932  7.368660
## DUSP1    5.083793  7.610593  7.631311  6.518594  5.059087  4.855161
## MMP10    3.252213  3.288078  3.264168  3.217279  3.377575  3.365157
##         CLL17.CEL CLL18.CEL CLL19.CEL CLL20.CEL CLL21.CEL CLL22.CEL
## MAPK3    5.325348  4.826131  5.212387  5.285830  5.581859  6.251678
## TIE1     2.350796  2.325163  2.432635  2.256547  2.348389  2.263849
## CYP2C19  3.502440  3.394410  3.617099  3.279726  3.391734  3.306811
## CXCR5    6.456285  6.824862  7.304803  8.757756  6.695295  7.372185
## DUSP1    5.176975  4.874563  4.097580  9.250585  7.657463  7.683677
## MMP10    3.451987  3.392632  3.574332  3.249886  3.269541  3.213493
##         CLL23.CEL CLL24.CEL CLL2.CEL CLL3.CEL CLL4.CEL CLL5.CEL CLL6.CEL
## MAPK3    5.480752  5.216033 5.966942 5.397508 5.281720 5.414718 5.460626
## TIE1     2.264434  2.344079 2.350073 2.406846 2.341961 2.372928 2.356978
## CYP2C19  3.341444  3.798335 3.427736 3.453564 3.412944 3.411922 3.396466
## CXCR5    7.616749  6.839187 7.545673 8.802729 7.307752 7.491507 8.063202
## DUSP1    8.700667  5.546996 9.611601 5.732182 6.483191 6.072558 9.919125
## MMP10    3.330000  3.545541 3.380044 3.426736 3.311255 3.411775 3.376011
##         CLL7.CEL CLL8.CEL CLL9.CEL
## MAPK3   5.897821 5.253883 5.214155
## TIE1    2.222276 2.254772 2.358544
## CYP2C19 3.247276 3.255148 3.365746
## CXCR5   7.014543 8.019108 7.432331
## DUSP1   5.149411 4.989604 5.339848
## MMP10   3.218438 3.271324 3.291604

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exp_mean <- apply(exp_sym, 1, mean)
exp_median <- apply(exp_sym,1, median)
exp_max <- apply(exp_sym, 1, max)            
exp_min <- apply(exp_sym, 1, min)
exp_sd <- apply(exp_sym, 1, sd)
exp_var <- apply(exp_sym, 1, var)
exp_md <- apply(exp_sym, 1, mad)
exp_sta <- data.frame(exp_mean, exp_median, exp_max, exp_min, exp_sd, exp_var, exp_md)
md_order <- exp_sta[order(exp_sta$exp_md, decreasing = T),]
md_order[1:50,1:5]
##           exp_mean exp_median   exp_max  exp_min    exp_sd
## FAM30A    5.708006   6.649724  9.901761 2.422295 2.7988082
## IGF2BP3   4.801569   4.659068  8.899667 2.335350 2.2955054
## DMD       6.791259   7.036325 10.653183 3.060286 2.5247064
## TCF7      7.665692   7.697969 10.976634 4.547559 2.1667457
## SLAMF1    5.385746   5.392873  8.385662 3.016306 1.9537439
## FOS       7.204708   7.009703 10.831309 3.581185 2.1980961
## LGALS1    6.612549   6.738502  9.275395 2.548286 2.0956955
## IGLC1    10.912833  10.695933 14.895714 6.516660 2.6146697
## ZAP70     5.395807   5.223290  7.581484 3.230575 1.5860361
## FCN1      5.549464   5.280685  9.321022 3.221669 1.8535899
## LHFPL2    5.880325   5.831304  8.627377 3.202529 1.7771356
## HBB      10.451111  10.835714 13.089985 3.556112 2.6459011
## S100A8    7.054067   7.125176  9.789312 3.976454 1.6721426
## GUSBP11   9.152709   9.136234 12.387608 6.296077 1.9078900
## COBLL1    6.307016   6.672768  8.602673 3.764972 1.7079344
## VIPR1     5.001816   4.783675  7.947431 3.013597 1.7173512
## PCDH9     7.564731   8.589865 10.379183 2.128020 2.8787582
## IGH       7.504659   7.615107  9.961620 4.058258 1.6876278
## ZNF804A   5.482021   4.933040  8.417146 3.489773 1.6262582
## TRIB2     5.582121   5.382651  8.275038 3.262417 1.6285227
## OAS1      5.781610   5.841878  7.812439 2.844634 1.5555384
## CCL3      4.955042   4.528829  8.200438 2.892649 1.6758945
## GNLY      5.758612   5.702251  7.874737 3.112976 1.4291897
## CYBB      5.465935   4.990295  7.925110 3.741134 1.5086211
## VAMP5     5.129844   5.224719  6.787208 3.168429 1.2736710
## RNASE6    7.502004   7.475790  9.761536 4.561619 1.3692587
## RGS2      8.665894   8.690715 11.060867 5.593852 1.6673174
## PLXNC1    6.152678   6.011995  9.936706 4.282411 1.4519185
## CAPG      7.147032   7.287538  9.311853 4.295201 1.3599095
## RBM38     5.625046   5.984977  7.712013 2.364198 1.5115862
## VCAN      5.724099   5.551630  9.690421 3.425727 1.6784676
## APBB2     5.028141   4.991633  7.223220 3.534210 1.2951444
## ARF6      9.261574   9.274190 11.084527 7.417333 1.1470937
## TGFBI     7.719464   8.355828 10.336590 3.705677 2.1598336
## NR4A2     5.332218   4.905362 10.162592 3.535849 1.7782290
## S100A9    5.283652   5.121900  9.274989 3.360444 1.5913846
## ZNF266    9.162509   8.963549 10.825413 7.465138 1.1059916
## TSPYL2    5.334349   4.961360  9.606263 3.519564 1.5168915
## CLEC2B    6.703088   6.200192 10.689725 4.346883 1.6435564
## FLNA      7.261863   7.427031  8.892456 4.910564 1.2846904
## H1FX      9.481071   9.371750 11.448482 6.315160 1.4222048
## DUSP5     5.707280   5.662890  8.399445 3.096216 1.4659308
## DUSP6     6.220190   6.163704  9.349607 3.924092 1.6169735
## ANXA4     6.285188   6.575309  8.848398 3.290936 1.6485526
## LPL       5.451203   4.535271  8.736220 3.497206 1.8272643
## THEMIS2   7.060804   6.925227  8.676046 5.638975 0.9956062
## P2RY14    5.503758   5.449599  9.268856 2.776078 1.7700218
## ARHGAP44  5.823372   5.937855  8.171374 3.285610 1.3502894
## TNFSF9    5.038986   4.935596  7.080714 2.827961 1.2324676
## PFN2      5.427243   5.891385  7.621100 2.987904 1.3889489

13提取基因的表达矩阵子集，绘制热图

根据第12步骤得到top 50 mad值的基因列表来取表达矩阵的子集，并且热图可视化子表达矩阵。试试看其它5种热图的包的不同效果。

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names_50 <- rownames(md_order[1:50,])
exp_mad_top50 <- exp_sym[match(names_50, rownames(exp_sym)),]
pheatmap::pheatmap(log2(exp_mad_top50))

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pheatmap::pheatmap(exp_mad_top50, scale=c("row"))

14取N个指标的交集并可视化：

UpSetR包 取不同统计学指标mean,median,max,mean,sd,var,mad的各top50基因列表， 使用UpSetR包来看他们之间的overlap情况

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mean_50 <- head(sort(exp_mean, decreasing = T),50)
median_50 <- head(sort(exp_median, decreasing = T),50)

写函数完成上述重复问题问题

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sta_50 <- function(x){
  x <- head(sort((apply(exp_sym, 1, x)), decreasing = T),50)
  x_50 <- x
  return(names((x_50)))
}
library('UpSetR')

取不含重复的并集

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sta_50_all <- unique(c(sta_50(mean), sta_50(median),sta_50(max),sta_50(min),sta_50(sd),sta_50(var), sta_50(mad)))

转成upset需要的格式，也就是1和0分别代表

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u_mean <- ifelse(sta_50_all %in% sta_50(mean), 1, 0)

再写个函数

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upset_sta <- function(x){
   x <- ifelse(sta_50_all %in% sta_50(x), 1, 0)
   u_x <- x
   return(u_x)
}
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upset_all <- data.frame(sta_50_all, upset_sta(mean),upset_sta(median),
                        upset_sta(max),upset_sta(min),upset_sta(sd),upset_sta(var),upset_sta(mad))
?upset
upset(upset_all, nsets = 7, matrix.color = 'black',main.bar.color = 'green',
      sets.bar.color = 'red',point.size = 2, line.size = 0.8,
      shade.color = 'red', matrix.dot.alpha = 0.5)

15提取表型数据

在第二步的基础上面提取CLL包里面的data(sCLLex) 数据对象的样本的表型数据。

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data("sCLLex")
pData(sCLLex)
##           SampleID  Disease
## CLL11.CEL    CLL11 progres.
## CLL12.CEL    CLL12   stable
## CLL13.CEL    CLL13 progres.
## CLL14.CEL    CLL14 progres.
## CLL15.CEL    CLL15 progres.
## CLL16.CEL    CLL16 progres.
## CLL17.CEL    CLL17   stable
## CLL18.CEL    CLL18   stable
## CLL19.CEL    CLL19 progres.
## CLL20.CEL    CLL20   stable
## CLL21.CEL    CLL21 progres.
## CLL22.CEL    CLL22   stable
## CLL23.CEL    CLL23 progres.
## CLL24.CEL    CLL24   stable
## CLL2.CEL      CLL2   stable
## CLL3.CEL      CLL3 progres.
## CLL4.CEL      CLL4 progres.
## CLL5.CEL      CLL5 progres.
## CLL6.CEL      CLL6 progres.
## CLL7.CEL      CLL7 progres.
## CLL8.CEL      CLL8 progres.
## CLL9.CEL      CLL9   stable

16对表达矩阵聚类画图添加表型数据

对所有样本的表达矩阵进行聚类并且绘图，然后添加样本的临床表型数据信息(更改样本名) #前面

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head(exp_sym)
##         CLL11.CEL CLL12.CEL CLL13.CEL CLL14.CEL CLL15.CEL CLL16.CEL
## MAPK3    5.743132  6.219412  5.523328  5.340477  5.229904  4.920686
## TIE1     2.285143  2.291229  2.287986  2.295313  2.662170  2.278040
## CYP2C19  3.309294  3.318466  3.354423  3.327130  3.365113  3.568353
## CXCR5    7.544884  7.671801  7.474025  7.152482  6.902932  7.368660
## DUSP1    5.083793  7.610593  7.631311  6.518594  5.059087  4.855161
## MMP10    3.252213  3.288078  3.264168  3.217279  3.377575  3.365157
##         CLL17.CEL CLL18.CEL CLL19.CEL CLL20.CEL CLL21.CEL CLL22.CEL
## MAPK3    5.325348  4.826131  5.212387  5.285830  5.581859  6.251678
## TIE1     2.350796  2.325163  2.432635  2.256547  2.348389  2.263849
## CYP2C19  3.502440  3.394410  3.617099  3.279726  3.391734  3.306811
## CXCR5    6.456285  6.824862  7.304803  8.757756  6.695295  7.372185
## DUSP1    5.176975  4.874563  4.097580  9.250585  7.657463  7.683677
## MMP10    3.451987  3.392632  3.574332  3.249886  3.269541  3.213493
##         CLL23.CEL CLL24.CEL CLL2.CEL CLL3.CEL CLL4.CEL CLL5.CEL CLL6.CEL
## MAPK3    5.480752  5.216033 5.966942 5.397508 5.281720 5.414718 5.460626
## TIE1     2.264434  2.344079 2.350073 2.406846 2.341961 2.372928 2.356978
## CYP2C19  3.341444  3.798335 3.427736 3.453564 3.412944 3.411922 3.396466
## CXCR5    7.616749  6.839187 7.545673 8.802729 7.307752 7.491507 8.063202
## DUSP1    8.700667  5.546996 9.611601 5.732182 6.483191 6.072558 9.919125
## MMP10    3.330000  3.545541 3.380044 3.426736 3.311255 3.411775 3.376011
##         CLL7.CEL CLL8.CEL CLL9.CEL
## MAPK3   5.897821 5.253883 5.214155
## TIE1    2.222276 2.254772 2.358544
## CYP2C19 3.247276 3.255148 3.365746
## CXCR5   7.014543 8.019108 7.432331
## DUSP1   5.149411 4.989604 5.339848
## MMP10   3.218438 3.271324 3.291604

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group_list <- as.character(pData(sCLLex)[,2])
colnames(exp_sym) <- paste(group_list, 1:22, sep = '')
head(exp_sym)
##         progres.1  stable2 progres.3 progres.4 progres.5 progres.6
## MAPK3    5.743132 6.219412  5.523328  5.340477  5.229904  4.920686
## TIE1     2.285143 2.291229  2.287986  2.295313  2.662170  2.278040
## CYP2C19  3.309294 3.318466  3.354423  3.327130  3.365113  3.568353
## CXCR5    7.544884 7.671801  7.474025  7.152482  6.902932  7.368660
## DUSP1    5.083793 7.610593  7.631311  6.518594  5.059087  4.855161
## MMP10    3.252213 3.288078  3.264168  3.217279  3.377575  3.365157
##          stable7  stable8 progres.9 stable10 progres.11 stable12
## MAPK3   5.325348 4.826131  5.212387 5.285830   5.581859 6.251678
## TIE1    2.350796 2.325163  2.432635 2.256547   2.348389 2.263849
## CYP2C19 3.502440 3.394410  3.617099 3.279726   3.391734 3.306811
## CXCR5   6.456285 6.824862  7.304803 8.757756   6.695295 7.372185
## DUSP1   5.176975 4.874563  4.097580 9.250585   7.657463 7.683677
## MMP10   3.451987 3.392632  3.574332 3.249886   3.269541 3.213493
##         progres.13 stable14 stable15 progres.16 progres.17 progres.18
## MAPK3     5.480752 5.216033 5.966942   5.397508   5.281720   5.414718
## TIE1      2.264434 2.344079 2.350073   2.406846   2.341961   2.372928
## CYP2C19   3.341444 3.798335 3.427736   3.453564   3.412944   3.411922
## CXCR5     7.616749 6.839187 7.545673   8.802729   7.307752   7.491507
## DUSP1     8.700667 5.546996 9.611601   5.732182   6.483191   6.072558
## MMP10     3.330000 3.545541 3.380044   3.426736   3.311255   3.411775
##         progres.19 progres.20 progres.21 stable22
## MAPK3     5.460626   5.897821   5.253883 5.214155
## TIE1      2.356978   2.222276   2.254772 2.358544
## CYP2C19   3.396466   3.247276   3.255148 3.365746
## CXCR5     8.063202   7.014543   8.019108 7.432331
## DUSP1     9.919125   5.149411   4.989604 5.339848
## MMP10     3.376011   3.218438   3.271324 3.291604

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t.exp <- t(exp_sym)
t.exp[1:5,1:5]
##              MAPK3     TIE1  CYP2C19    CXCR5    DUSP1
## progres.1 5.743132 2.285143 3.309294 7.544884 5.083793
## stable2   6.219412 2.291229 3.318466 7.671801 7.610593
## progres.3 5.523328 2.287986 3.354423 7.474025 7.631311
## progres.4 5.340477 2.295313 3.327130 7.152482 6.518594
## progres.5 5.229904 2.662170 3.365113 6.902932 5.059087

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hc <- hclust(dist(t.exp))
plot(as.dendrogram(hc))

用factoextra包画

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exp_cluster <- t(exp_sym)
exp_clust_dist <- dist(exp_cluster, method = 'euclidean')
hc <- hclust(exp_clust_dist,'ward.D')
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library(factoextra)
## Welcome! Related Books: `Practical Guide To Cluster Analysis in R` at https://goo.gl/13EFCZ

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fviz_dend(hc, k=4, cex = 0.5,
          k_colors = c("#2E9FDF", "#00AFBB", "#E7B800", "#FC4E07"),
          color_labels_by_k = TRUE, rect = TRUE)

17对所有样本PCA分析添加表型信息

对所有样本的表达矩阵进行PCA分析并且绘图，同样要添加表型信息

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library(devtools)
install_github('sinhrks/ggfortify')
## Skipping install of 'ggfortify' from a github remote, the SHA1 (107768f6) has not changed since last install.
##   Use `force = TRUE` to force installation

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library(ggfortify)
df <- as.data.frame(t(exp_sym))
df$group <- group$Disease
autoplot(prcomp(df[,1:(ncol(df)-1)]), data=df, 
         label = TRUE, colour = 'group')

18所有样本表达矩阵批量T检验

根据表达矩阵及样本分组信息进行批量T检验，得到检验结果表格

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dat <- exp_sym
group_list <- as.factor(group_list)
group1 <- which(group_list == levels(group_list)[1])
group2 <- which(group_list == levels(group_list)[2])
dat1 <- dat[,group1]
dat2 <- dat[,group2]
dat <- cbind(dat1, dat2)
pvals <- apply(dat, 1, function(x){
  t.test(as.numeric(x)~group_list)$p.value
})
head(pvals)
##      MAPK3       TIE1    CYP2C19      CXCR5      DUSP1      MMP10 
## 0.65221728 0.32547921 0.10930650 0.52972986 0.11181740 0.08771106

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p.adj <- p.adjust(pvals, method = 'BH')
avg_1 <- log2(rowMeans(dat1))
## Warning: NaNs produced

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avg_2 <- log2(rowMeans(dat2))
## Warning: NaNs produced

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log2FC <- avg_2-avg_1
deg_t.test <- cbind(avg_1, avg_2, log2FC, pvals, p.adj)
deg_t.test <- deg_t.test[order(deg_t.test[,4]),]
head(deg_t.test)
##           avg_1    avg_2      log2FC        pvals     p.adj
## ST14   2.491256 2.364128 -0.12712815 0.0001236326 0.4156259
## LTB    2.961047 2.807431 -0.15361585 0.0002103122 0.4156259
## CORO1A 3.239158 3.149793 -0.08936553 0.0002370691 0.4156259
## PPM1D  2.356110 2.425399  0.06928926 0.0004783848 0.4156259
## ORAI2  2.459287 2.199910 -0.25937653 0.0005244971 0.4156259
## GNAI2  3.319264 3.275868 -0.04339579 0.0005434369 0.4156259

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class(deg_t.test)
## [1] "matrix"

19limma包进行DEGs分析

使用limma包对表达矩阵及样本分组信息进行差异分析，得到差异分析表格，

重点看logFC和P值，画个火山图(就是logFC和-log10(P值)的散点图)。

19.1 构建设计矩阵design matrix

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library(limma)
## 
## Attaching package: 'limma'

## The following object is masked from 'package:BiocGenerics':
## 
##     plotMA

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design <- model.matrix(~0+factor(group_list))
colnames(design) <- levels(factor(group_list))
rownames(design) <- colnames(exp_sym)
design
##            progres. stable
## progres.1         1      0
## stable2           0      1
## progres.3         1      0
## progres.4         1      0
## progres.5         1      0
## progres.6         1      0
## stable7           0      1
## stable8           0      1
## progres.9         1      0
## stable10          0      1
## progres.11        1      0
## stable12          0      1
## progres.13        1      0
## stable14          0      1
## stable15          0      1
## progres.16        1      0
## progres.17        1      0
## progres.18        1      0
## progres.19        1      0
## progres.20        1      0
## progres.21        1      0
## stable22          0      1
## attr(,"assign")
## [1] 1 1
## attr(,"contrasts")
## attr(,"contrasts")$`factor(group_list)`
## [1] "contr.treatment"

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fit <- lmFit(exp_sym, design)

19.2 构建比较矩阵

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contrast.matrix <- makeContrasts(paste0(unique(group_list), 
                            collapse = '-'), levels = design)
fit2 <- contrasts.fit(fit, contrast.matrix)
fit3 <- eBayes(fit2)

19.3差异表达矩阵获取

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mtx <- topTable(fit3, coef = 1, n=Inf)
DEG_mtx <- na.omit(mtx)
dim(mtx)
## [1] 8585    6

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dim(DEG_mtx)
## [1] 8585    6

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head(DEG_mtx)
##              logFC  AveExpr         t      P.Value  adj.P.Val        B
## TBC1D2B -1.0284628 5.620700 -5.837412 8.241439e-06 0.02237023 3.351806
## CLIC1    0.9888221 9.954273  5.772861 9.560444e-06 0.02237023 3.230776
## DLEU1    1.8301554 6.950685  5.740842 1.029277e-05 0.02237023 3.170506
## SH3BP2  -1.3835699 4.463438 -5.735395 1.042294e-05 0.02237023 3.160238
## GPM6A    2.5471980 6.915045  5.043131 5.269777e-05 0.08731675 1.821536
## YTHDC2  -0.5187135 7.602354 -4.873850 7.879310e-05 0.08731675 1.485255

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#write.csv(DEG_mtx,"DEG_mtx.csv",quote = F)

19.4火山图

第一步，设定阈值

界定上调下调和不表达的基因

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DEG <- DEG_mtx
logFC_cutoff <- with(DEG, mean(abs(logFC))+2*sd(abs(logFC)))
DEG$change = as.factor(ifelse(DEG$P.Value < 0.05 & abs(DEG$logFC) > logFC_cutoff,
                              ifelse(DEG$logFC > logFC_cutoff ,'UP','DOWN'),'NOT')
)

这是两个ifelse判断嵌套。先了解ifelse的结构，ifelse（条件，yes，no），如果满足条件，那么返回yes/或者执行yes所处的下一个命令；反之返回no 这里外层的ifelse中DEG$P.Value < 0.05 & abs(DEG$logFC) > logFC_cutoff是判断条件，这个就是看p值和logFC是不是达到了他们设定的阈值【p是0.05，logFC是logFC_cutoff】，如果达到了就进行下一个ifelse，达不到就返回NOT； 第二层ifelse也是上来一个条件：DEG$logFC > logFC_cutoff，如果达到了，就返回UP即上调基因，达不到就是下调DOWN 最后将判断结果转位因子型，得到DOWN、UP、NOT的三种因子

第二步，设定火山图标题

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this_tile <- paste0('Cutoff for logFC is ',round(logFC_cutoff,3),
                    '\nThe number of up gene is ',nrow(DEG[DEG$change =='UP',]) ,
                    '\nThe number of down gene is ',nrow(DEG[DEG$change =='DOWN',])
)

第三步，画图

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g = ggplot(data=DEG, aes(x=logFC, y=-log10(P.Value), color=change)) +
  geom_point(alpha=0.4, size=1.75) +
  theme_set(theme_set(theme_bw(base_size=20)))+
  xlab("log2 fold change") + ylab("-log10 p-value") +
  ggtitle( this_tile ) + theme(plot.title = element_text(size=15,hjust = 0.5))+
  scale_colour_manual(values = c('blue','black','red')) ## corresponding to the levels(res$change)
print(g)

第四步，画个漂亮的图

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P_volcano=ggplot(DEG,aes(x=logFC,y=-log10(P.Value)))+
  geom_point(aes(color=change))+
  #设置点的颜色
  scale_color_manual(values =c("UP" = "red", "DOWN" = "blue", "NOT" = "grey"))+
  labs(x="log2FC",y="-log10FDR")+
  #增加阈值线:分别对应FDR=0.05,|log2FC|=1
  geom_hline(yintercept=-log10(0.05),linetype=4)+
  geom_vline(xintercept=c(-1,1),linetype=4)+
  xlim(-3,3)+
  theme(plot.title = element_text(size = 25,face = "bold", vjust = 0.5, hjust = 0.5),
        legend.title = element_blank(),
        legend.text = element_text(size = 18, face = "bold"),
        legend.position = 'right',
        legend.key.size=unit(0.8,'cm'),
        axis.ticks.x=element_blank(),
        axis.text.x=element_text(size = 15,face = "bold", vjust = 0.5, hjust = 0.5),
        axis.text.y=element_text(size = 15,face = "bold", vjust = 0.5, hjust = 0.5),
        axis.title.x = element_text(size = 20,face = "bold", vjust = 0.5, hjust = 0.5),
        axis.title.y = element_text(size = 20,face = "bold", vjust = 0.5, hjust = 0.5),
        panel.background = element_rect(fill = "transparent",colour = "black"),
        panel.grid.minor = element_blank(),
        panel.grid.major = element_blank(),
        plot.background = element_rect(fill = "transparent",colour = "black"))
P_volcano

20T检验结果和limma结果比较

对T检验结果的P值和limma包差异分析的P值画散点图，看看哪些基因相差很大

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deg_t.test <- as.data.frame(deg_t.test)
t_limma_pval <- cbind(deg_t.test,DEG)[,c(4,9)]
head(t_limma_pval)
##               pvals      P.Value
## ST14   0.0001236326 8.241439e-06
## LTB    0.0002103122 9.560444e-06
## CORO1A 0.0002370691 1.029277e-05
## PPM1D  0.0004783848 1.042294e-05
## ORAI2  0.0005244971 5.269777e-05
## GNAI2  0.0005434369 7.879310e-05

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plot(t_limma_pval)

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pheatmap::pheatmap(cor(t_limma_pval))


看哪些基因在两种方法种的p值差异比较大

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