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comparison RNAseqDataAnnotation/RNAseqDataAnnotation.R @ 26:f183f8648c5a draft default tip

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author eganrol
date Wed, 10 Dec 2014 06:42:21 -0500
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25:ea3f7a6504e0 26:f183f8648c5a
1 #Author : keime / lornage
2 #Date : 2014/11
3
4
5 ########################################################################################################
6 #This function concatenates htseq-count result files, normalizes data and annotates data using Ensembl annotations
7
8 #arguments
9 #Species : Name of the species
10 #ensversion : version of Ensembl to use
11 #fileout : tab-delimited file containing for each library ; gene id, raw read counts, normalized data as well as normalized data/gene length
12 #corresp : data.frame linking the file loaded into galaxy to the corresponding condition
13 #nfiles : number of files(conditions)
14
15 #output : a data.frame with the following columns :
16 #ensembl gene id
17 #raw read counts for each library (one column per library)
18 #normalized data for each library (one column per library)
19 #normalized data divided by gene length for each library (one column per library)
20 #Gene name
21 #Description
22
23 #require : biomaRt and DESeq2 Bioconductor packages / package plyr1.8.1
24
25 #Methods :
26 #Normalization is performed using the method described in Genome Biology 2010;11(10):R106
27 #and implemented in the DESeq2 Bioconductor package
28 #Gene length correspond to the median of the size of all transcripts corresponding to this gene
29 #########################################################################################################
30
31
32
33 RNAseqDataAnnotation = function(Species, ensversion, fileout, corresp ,nfiles){
34
35 #Create a string vector called libnames that contains the name of the samples
36 libnames=rep("",nfiles)
37 for (i in 1:nfiles){
38 libnames[i]=toString(corresp$Sample_name[i])
39 }
40
41 #For all files in corresp read the corresponding file into R
42 suppressPackageStartupMessages(library(plyr, lib.loc = NULL, character.only = FALSE, logical.return = FALSE, warn.conflicts = FALSE, verbose=FALSE, quietly = TRUE))
43 datalist = list()
44 for(i in 1:nfiles){
45 rawdata=read.table(toString(corresp$Files[i]), header =T, sep ="\t")
46 #noSpikes_htseq.
47 nbrrows=nrow(rawdata)
48 datalist[[i]]=rawdata[1:(nbrrows-5), ] # skip the last 5 lines of HTSeq-count files
49 colnames(datalist[[i]]) = c("ID",libnames[i])
50 }
51
52 #Join all the files in a data.frame called datafile with rownames = gene id
53 datafile = join_all(datalist, by = "ID", type = "left", match = "all")
54
55 #Calculate the number of geneID pro file
56 nbID=data.frame(rep("",nfiles))
57 for(i in 1:nfiles){
58 nbID[,i]=nrow(datalist[[i]])
59 }
60 totalnbID=apply((nbID[,1:nfiles]),1,sum)
61
62 #Verify that all the files contain the same gene ID
63 if (nrow(datafile)*nfiles==totalnbID[1]){
64
65 #Suppress genes not expressed in all samples
66 datafile = datafile[apply(datafile[,2:(nfiles+1)],1,sum)!=0,]
67 row.names(datafile)=datafile[,1]
68 data=datafile[,-1]
69
70 #Number of libraries
71 nblib= dim(data)[2]
72 #Determine Data + normalization if the specie is not known
73 if (Species=="None"){
74 #Normalized data calculation
75 nbcol = dim(data)[2] #nb of column in the data.frame
76 suppressPackageStartupMessages(library(DESeq2, lib.loc = NULL, character.only = FALSE, logical.return = FALSE, warn.conflicts = FALSE, verbose=FALSE, quietly = TRUE))
77 conds = factor(1:nblib)
78 design = data.frame(Condition=conds)
79 dds = DESeqDataSetFromMatrix(countData=data, colData=design, design=~Condition)
80 dds = estimateSizeFactors(dds)
81 datanorm = t(t(data)/sizeFactors(dds))
82
83 #Data + normalization
84 dataall = data.frame(row.names(datafile), data, datanorm )
85
86 #Renames columns
87 colnames(dataall) = c("Ensembl gene id", paste(libnames,"(raw read counts)"), paste(libnames,"(normalized)"))
88 write.table(dataall, file=fileout, sep="\t", quote=F, row.names=F)
89 }
90
91 #Determine Data + normalization + annotation if the specie is known
92 else{
93 #Add annotations and calculate gene length
94 suppressPackageStartupMessages(library(biomaRt, lib.loc = NULL, character.only = FALSE, logical.return = FALSE, warn.conflicts = FALSE,verbose=FALSE, quietly = TRUE))
95
96 #Convert Ensembl version to host
97 correspondingdate = toString(ensversion)
98 host = paste(correspondingdate, ".archive.ensembl.org/biomart/martservice/", sep="")
99
100 #Load the correct bmdataset
101 bmdataset = toString(Species)
102 ensembl=useMart("ENSEMBL_MART_ENSEMBL", host=host, dataset=bmdataset)
103 if (toString(ensversion)=="oct2014" | toString(ensversion)=="aug2014" ) {
104 annotation1 = getBM(attributes=c("ensembl_gene_id","external_gene_name","description", "ensembl_transcript_id","exon_chrom_start","exon_chrom_end"),filters="ensembl_gene_id", values=rownames(data), mart=ensembl)
105 }
106 else{
107 annotation1 = getBM(attributes=c("ensembl_gene_id","external_gene_id","description", "ensembl_transcript_id","exon_chrom_start","exon_chrom_end"),filters="ensembl_gene_id", values=rownames(data), mart=ensembl)
108 }
109
110 #because all the annotations are not always found in a first step
111 not = rownames(data)[!rownames(data) %in% unique(annotation1$ensembl_gene_id)]
112 if (length(not) !=0){
113 if (toString(ensversion)=="oct2014" | toString(ensversion)=="aug2014" ) {
114 annotationnot = getBM(attributes=c("ensembl_gene_id","external_gene_name","description", "ensembl_transcript_id","exon_chrom_start","exon_chrom_end"),filters="ensembl_gene_id", values=not, mart=ensembl)
115 annotation2 = rbind(annotation1, annotationnot)
116 }
117 else {
118 annotationnot = getBM(attributes=c("ensembl_gene_id","external_gene_id","description", "ensembl_transcript_id","exon_chrom_start","exon_chrom_end"), filters="ensembl_gene_id", values=not, mart=ensembl)
119 annotation2 = rbind(annotation1, annotationnot)
120 }
121 }
122 else{
123 annotation2 = annotation1
124 }
125
126
127 #because all the annotations are not always found in a first or second step
128 not = rownames(data)[!rownames(data) %in% unique(annotation2$ensembl_gene_id)]
129 if (length(not) !=0){
130 if (toString(ensversion)=="oct2014" | toString(ensversion)=="aug2014" ) {
131 annotationnot = getBM(attributes=c("ensembl_gene_id","external_gene_name","description", "ensembl_transcript_id","exon_chrom_start","exon_chrom_end"),filters="ensembl_gene_id", values=not, mart=ensembl)
132 annotation = rbind(annotation2, annotationnot)
133 }
134 else {
135 annotationnot = getBM(attributes=c("ensembl_gene_id","external_gene_id","description", "ensembl_transcript_id","exon_chrom_start","exon_chrom_end"), filters="ensembl_gene_id", values=not, mart=ensembl)
136 annotation = rbind(annotation2, annotationnot)
137 }
138 }
139 else{
140 annotation = annotation2
141 }
142
143 #Exon length
144 ensinfos.exlen = data.frame(annotation$ensembl_gene_id, annotation$ensembl_transcript_id, abs(annotation$exon_chrom_start - annotation$exon_chrom_end)+1)
145 colnames(ensinfos.exlen) = c("ensembl_gene_id", "ensembl_transcript_id", "exon_length")
146
147 #Transcript length
148 tlen = tapply(ensinfos.exlen$exon_length, ensinfos.exlen$ensembl_transcript_id, sum)
149 tlen.gene = merge(tlen, unique(ensinfos.exlen[,1:2]), by.x="row.names", by.y="ensembl_transcript_id")
150 colnames(tlen.gene) = c("ensembl_transcript_id", "transcript_length","ensembl_gene_id")
151
152 #Gene length = median of the size of all transcripts corresponding to this gene
153 glen = tapply(tlen.gene$transcript_length, tlen.gene$ensembl_gene_id, median)
154
155 #Data with gene length
156 datalen = merge(data, glen, by="row.names")
157 colnames(datalen) = c("Ensembl_gene_id",colnames(data), "Gene_length")
158
159 #Data with annotations and gene length
160 annotationgene = unique(annotation[,1:3])
161 dataannot = merge(datalen, annotationgene, by.x="Ensembl_gene_id", by.y="ensembl_gene_id")
162
163 #To keep only the first part of the gene description (before [)
164 tmpdesc = strsplit(as.character(dataannot$description),"[", fixed=T)
165 f = function(l){
166 if (length(l)>=1){
167 return(l[[1]])
168 }
169 else{
170 return("")
171 }
172 }
173 tmpdescok = unlist(lapply(tmpdesc, f))
174 dataannot$description = tmpdescok
175
176 #Normalized data calculation
177 nbcol = dim(dataannot)[2] #nb of column in the data.frame
178 suppressPackageStartupMessages(library(DESeq2, lib.loc = NULL, character.only = FALSE, logical.return = FALSE, warn.conflicts = FALSE,verbose=FALSE, quietly = TRUE))
179 conds = factor(1:nblib)
180 design = data.frame(Condition=conds)
181 dds = DESeqDataSetFromMatrix(countData=dataannot[,-c(1,nbcol,nbcol-1,nbcol-2)], colData=design, design=~Condition)
182 dds = estimateSizeFactors(dds)
183 datanorm = t(t(dataannot[,-c(1,nbcol,nbcol-1,nbcol-2)])/sizeFactors(dds))
184
185 #Normalized data adjusted for gene length (normalized data / gene length)
186 rpkn = datanorm / (as.vector(dataannot[,nbcol-2]/1000 ))
187
188 #Data + annotations + rpkn
189 dataall = data.frame(dataannot[,-c(nbcol,nbcol-1,nbcol-2)] , datanorm, rpkn, dataannot[,c(nbcol-1,nbcol)] )
190
191 #Renames columns
192 colnames(dataall) = c("Ensembl gene id", paste(libnames,"(raw read counts)"), paste(libnames,"(normalized)"), paste(libnames,"(normalized and divided by gene length in kb)"), "Gene name", "Description")
193 write.table(dataall, file=fileout, sep="\t", quote=F, row.names=F)
194
195 }
196 }
197 else{
198 print("The files are not the same length")
199 }
200 }
201
202 # Build a dataframe containing the files loaded into galaxy and their corresponding condition
203
204 args <- commandArgs(trailingOnly = TRUE)
205
206 Files=c()
207 Sample_name =c()
208 nbcells = (length(args)-3)
209 for (i in seq(1,nbcells,2)){
210 Files = c(Files, args[3+i])
211 Sample_name = c(Sample_name, args[4+i])
212 }
213 nfiles=nbcells/2
214 corresp = data.frame(Files=Files, Sample_name=Sample_name)
215
216 # Take the informations given by the galaxy user to run the script
217 RNAseqDataAnnotation(args[1], args[2],args[3],corresp,nfiles)
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