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files /dev/null and b/analysis.MS/differentialAnalysisGraphs/padjDistribution.pdf differ diff --git a/analysis.MS/differentialAnalysisGraphs/pvalDistribution.pdf b/analysis.MS/differentialAnalysisGraphs/pvalDistribution.pdf new file mode 100644 index 0000000..ee5d568 Binary files /dev/null and b/analysis.MS/differentialAnalysisGraphs/pvalDistribution.pdf differ diff --git a/analysis.MS/differentialAnalysisGraphs/varianceEstimation.pdf b/analysis.MS/differentialAnalysisGraphs/varianceEstimation.pdf new file mode 100644 index 0000000..634375e Binary files /dev/null and b/analysis.MS/differentialAnalysisGraphs/varianceEstimation.pdf differ diff --git a/analysis.MS/report b/analysis.MS/report new file mode 100644 index 0000000..04a6dc9 --- /dev/null +++ b/analysis.MS/report @@ -0,0 +1,55 @@ +1.Download manually the 4 datasets +ERR990557 +ERR990558 +ERR990559 +ERR990560 + +2.Download fastq files with a bash script downloadFastq.sh +To run it $ bash downloadFastq.sh repertoryOfDatasets + +3.Unzip manually fastq files +ERR990557.fastq +ERR990558.fastq +ERR990559.fastq +ERR990560.fastq + +4.Select the first 8 000 000 reads in each fastq files with a python script selectReads.py +Creation of sample files +ERR990557s.fastq +ERR990558s.fastq +ERR990559s.fastq +ERR990560s.fastq +The principal fonction fonction selectReadForAllFiles takes the list of fastq files and the number of reads wanted in each sample files + +5.Align reads datasets to reference genomes with bowtie +Prerequisite : download Drosophila melanogaster index and unzip it +Use default parameters +To run bowtie +$ ./bowtie -S d_melanogaster_fb5_22 fastqFile >outputFile +Creation of sam files +ERR990557s.bowtie.sam +ERR990558s.bowtie.sam +ERR990559s.bowtie.sam +ERR990560s.bowtie.sam + +6.Count reads aligning to genome's genes with HTSeq +Prerequisite : download and unzip gtf file of Drosophila melanogaster +To run HTSeq +$ python -m HTSeq.scripts.count samFile gtfFile >outputFile +Creation of 4 files, folder countFiles with theses files given +ERR990557s.count.txt +ERR990558s.count.txt +ERR990559s.count.txt +ERR990560s.count.txt + +7.Differential analysis with DESeq2 package on R +Script differentialAnalysis.R +Graphs reporting analysis in folder differentialAnalysisGraphs +file with differentially expressed gene resultOfDifferantialAnalysis.txt + + + + + + + diff --git a/analysis.MS/resultOfDifferantialAnalysis.txt b/analysis.MS/resultOfDifferantialAnalysis.txt new file mode 100644 index 0000000..91882f2 --- /dev/null +++ b/analysis.MS/resultOfDifferantialAnalysis.txt @@ -0,0 +1,10 @@ +baseMean log2FoldChange lfcSE stat pvalue padj +FBgn0003892 273.650858940795 4.22489167801591 0.748709288961954 5.64290004184869 1.67209434203519e-08 3.04878535031083e-05 +FBgn0003961 718.730743284043 3.68326480073617 0.722672951426683 5.0967243114119 3.45581026526622e-07 0.000472582053775155 +FBgn0031523 148.98239886426 3.2679796419709 0.742188225063946 4.40316826865495 1.06681357069062e-05 0.00680675265212847 +FBgn0032049 241.050037802806 5.13852162101445 0.668717526285134 7.68414378124649 1.54023606280711e-14 8.42509126355492e-11 +FBgn0032235 73.7391147520904 3.73481514906243 0.754057228824972 4.95295981033468 7.30930754341963e-07 0.000799638245250107 +FBgn0032505 107.722750765249 4.08443972447685 0.710725067842802 5.74686318139019 9.09142342257462e-09 2.48650430607416e-05 +FBgn0032913 702.593594885596 3.12529718041409 0.71148858967721 4.39261743021345 1.11994102137397e-05 0.00680675265212847 +FBgn0033354 143.433506890372 3.41694304832748 0.737368530292016 4.63396918630944 3.5872041293664e-06 0.00327033443127237 +FBgn0035343 225.528304796664 3.32562295524925 0.737523134797493 4.50917781197792 6.50793470713763e-06 0.00508548612114897 diff --git a/analysis.MS/scripts/differentialAnalysis.R b/analysis.MS/scripts/differentialAnalysis.R new file mode 100644 index 0000000..60c4e6e --- /dev/null +++ b/analysis.MS/scripts/differentialAnalysis.R @@ -0,0 +1,48 @@ +#Rscript for differential analysis + +library(DESeq2) + +# DESeqDataSet object creation +dir="/home/mariam/Documents/countFiles" +HTSeqCountFiles = c("ERR990557s.count.txt", "ERR990558s.count.txt", "ERR990559s.count.txt", "ERR990560s.count.txt") +stages = c("1", "2","2","2") +samples = c("ERR990557", "ERR990558", "ERR990559", "ERR990560") +samplesTable = data.frame(sampleName=samples, fileName=HTSeqCountFiles, condition=stages) +dds = DESeqDataSetFromHTSeqCount(sampleTable=samplesTable, directory=dir, design= ~ condition) + +# normalization of counts + +# calculation of sizeFactors +dds = estimateSizeFactors(dds) +sizeFactors(dds) + +# bar plots +barplot(colSums(counts(dds)),main='number of reads mapped on genes for each experiment') + +# boxplots +boxplot(log2(counts(dds)+1),main='Barplot which represent logfc dispersion for each experiment\nbefore normalization') +boxplot(log2(counts(dds,normalized=TRUE)+1),main='Barplot which represent logfc dispersion for each experiment\nafter normalization') + +# variance estimations +dds = estimateDispersions(dds) +plotDispEsts(dds,main='Representation of variance') + +# differential analysis +dds = nbinomWaldTest(dds) +res = results(dds) + +# diagnostic plots + +# MA plot +plotMA(res,main='MA plot for differential analysis') + +# Histogram of the p values +hist(res$pvalue,main='distribution of p-values',breaks=20,col="grey") +hist(res$padj,main='distribution of adjusted p-values',breaks=20,col="blue") + +# selection of the genes with an adjusted p-value<0.01 +ind=which(res$padj<0.01) +res=as.matrix(res) +print(res) +res = res[ind,] +write.table(res, "resultOfDifferentialAnalysis.txt", row.name=T, quote=F, sep='\t') diff --git a/analysis.MS/scripts/downloadFastq.sh b/analysis.MS/scripts/downloadFastq.sh new file mode 100644 index 0000000..b5ec2a9 --- /dev/null +++ b/analysis.MS/scripts/downloadFastq.sh @@ -0,0 +1,24 @@ +export repertory=$1 + +files=$(ls $repertory) +echo $files +cd $repertory +chaine="gz" +see='nothing' + +for file in $files; +do + for line in $(<$file) + do + IFS='/' read a b c d e f<<< "$line" + IFS='.' read g h i<<< "$f" + if [ "$i" == "$chaine" ] && [ "$see" != "$line" ] + then + echo $i + wget $line + see=$line + + fi + + done +done diff --git a/analysis.MS/scripts/selectReads.py b/analysis.MS/scripts/selectReads.py new file mode 100644 index 0000000..f68175d --- /dev/null +++ b/analysis.MS/scripts/selectReads.py @@ -0,0 +1,44 @@ +# -*- coding: utf-8 -*- +""" +Created on Wed Sep 27 17:40:23 2017 + +@author: mariam +""" +import re + +def seletReads(fileRead,number,fileWrite,name): + count=0 + fi=open(fileRead,'r') + fiWrite=open(fileWrite,'w') + line=fi.readline() + while(1): + + if line[0]=='+' and len(line)==2: + count+=1 + if count==number: + fiWrite.write(line) + line=fi.readline() + fiWrite.write(line) + break; + fiWrite.write(line) + line=fi.readline() + fi.close() + fiWrite.close() + return + +def selectReadForAllFiles(filesNames,number): + for i in filesNames: + print i + fileFin=i.split('/') + fileFin=fileFin[len(fileFin)-1] + fileFin=fileFin.split('.') + name=fileFin[0] + fileFin[0]=fileFin[0]+'s' + fileFin='.'.join(fileFin) + seletReads(i,number,fileFin,name) + return + +files=['/home/mariam/Documents/datasets/ERR990557.fastq','/home/mariam/Documents/datasets/ERR990558.fastq','/home/mariam/Documents/datasets/ERR990559.fastq','/home/mariam/Documents/datasets/ERR990560.fastq'] +selectReadForAllFiles(files,8000000) + + \ No newline at end of file