PRJNA297798

Guillermo Ayala

2024-04-02

Data

This data has been analyzed in
>Kaletsky, R.; Lakhina, V.; Arey, R.; Williams, A.; Landis, J.; Ashraf, J. & Murphy, C. T. The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators Nature, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016, 529, 92-96

Download the sra files

wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433703/SRR2969230/SRR2969230.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433714/SRR2969231/SRR2969231.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433717/SRR2969232/SRR2969232.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433729/SRR2969233/SRR2969233.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433729/SRR2979473/SRR2979473.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433737/SRR2979475/SRR2979475.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433737/SRR2969234/SRR2969234.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433838/SRR2979476/SRR2979476.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433838/SRR2969235/SRR2969235.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433860/SRR2969236/SRR2969236.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433862/SRR2969237/SRR2969237.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433865/SRR2969238/SRR2969238.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433869/SRR2969239/SRR2969239.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433873/SRR2969240/SRR2969240.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433875/SRR2969241/SRR2969241.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433876/SRR2969242/SRR2969242.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433877/SRR2969243/SRR2969243.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433878/SRR2969244/SRR2969244.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433879/SRR2969245/SRR2969245.sra
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX143/SRX1433884/SRR2969246/SRR2969246.sra

Generating the fastq files

fastq-dump -I --split-files SRR2969230.sra
fastq-dump -I --split-files SRR2969231.sra
fastq-dump -I --split-files SRR2969232.sra
fastq-dump -I --split-files SRR2969233.sra
fastq-dump -I --split-files SRR2969234.sra
fastq-dump -I --split-files SRR2969235.sra
fastq-dump -I --split-files SRR2969236.sra
fastq-dump -I --split-files SRR2969237.sra
fastq-dump -I --split-files SRR2969238.sra
fastq-dump -I --split-files SRR2969239.sra
fastq-dump -I --split-files SRR2969240.sra
fastq-dump -I --split-files SRR2969241.sra
fastq-dump -I --split-files SRR2969242.sra
fastq-dump -I --split-files SRR2969243.sra
fastq-dump -I --split-files SRR2969244.sra
fastq-dump -I --split-files SRR2969245.sra
fastq-dump -I --split-files SRR2969246.sra
fastq-dump -I --split-files SRR2979473.sra
fastq-dump -I --split-files SRR2979475.sra
fastq-dump -I --split-files SRR2979476.sra

Download the index file for bowtie2

wget ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz

Generating the directories with the index files

gzip -d Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz
tar xvf Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz

Short read alignment using bowtie2

bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -1 SRR2969230_1.fastq -2 SRR2969230_2.fastq -S  SRR2969230.sam
rm SRR2969230_*.fastq 
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -1 SRR2969231_1.fastq -2 SRR2969231_2.fastq -S  SRR2969231.sam
rm SRR2969231_*.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -1 SRR2969232_1.fastq -2 SRR2969232_2.fastq -S  SRR2969232.sam
rm SRR2969232_*.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969233_1.fastq -S SRR2969233.sam
rm SRR2969233_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969234_1.fastq -S SRR2969234.sam
rm SRR2969234_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969235_1.fastq -S SRR2969235.sam
rm SRR2969235_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -1 SRR2969236_1.fastq -2 SRR2969236_2.fastq -S  SRR2969236.sam
rm SRR2969236_*.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -1 SRR2969237_1.fastq -2 SRR2969237_2.fastq -S  SRR2969237.sam
rm SRR2969237_*.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -1 SRR2969238_1.fastq -2 SRR2969238_2.fastq -S  SRR2969238.sam
rm SRR2969238_*.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969239_1.fastq -S SRR2969239.sam
rm SRR2969239_1.fastq 
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969240_1.fastq -S SRR2969240.sam
rm SRR2969240_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969241_1.fastq -S SRR2969241.sam
rm SRR2969241_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969242_1.fastq -S SRR2969242.sam
rm SRR2969242_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969243_1.fastq -S SRR2969243.sam
rm SRR2969243_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969244_1.fastq -S SRR2969244.sam
rm SRR2969244_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969245_1.fastq -S SRR2969245.sam
rm SRR2969245_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2969246_1.fastq -S SRR2969246.sam
rm SRR2969246_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2979473_1.fastq -S SRR2979473.sam
rm SRR2979473_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2979475_1.fastq -S SRR2979475.sam
rm SRR2979475_1.fastq
bowtie2 -x Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome -U SRR2979476_1.fastq -S SRR2979476.sam
rm SRR2979476_1.fastq

From sam to bam using samtools and sorting the reads

samtools view -bS SRR2969230.sam | samtools sort - SRR2969230
rm SRR2969230.sam
samtools view -bS SRR2969231.sam | samtools sort - SRR2969231
rm SRR2969231.sam
samtools view -bS SRR2969232.sam | samtools sort - SRR2969232
rm SRR2969232.sam
samtools view -bS SRR2969233.sam | samtools sort - SRR2969233
rm SRR2969233.sam
samtools view -bS SRR2969234.sam | samtools sort - SRR2969234
rm SRR2969234.sam
samtools view -bS SRR2969235.sam | samtools sort - SRR2969235
rm SRR2969235.sam
samtools view -bS SRR2969236.sam | samtools sort - SRR2969236
rm SRR2969236.sam
samtools view -bS SRR2969237.sam | samtools sort - SRR2969237
rm SRR2969237.sam
samtools view -bS SRR2969238.sam | samtools sort - SRR2969238
rm SRR2969238.sam
samtools view -bS SRR2969239.sam | samtools sort - SRR2969239
rm SRR2969239.sam
samtools view -bS SRR2969240.sam | samtools sort - SRR2969240
rm SRR2969240.sam
samtools view -bS SRR2969241.sam | samtools sort - SRR2969241
rm SRR2969241.sam
samtools view -bS SRR2969242.sam | samtools sort - SRR2969242
rm SRR2969242.sam
samtools view -bS SRR2969243.sam | samtools sort - SRR2969243
rm SRR2969243.sam
samtools view -bS SRR2969244.sam | samtools sort - SRR2969244
rm SRR2969244.sam
samtools view -bS SRR2969245.sam | samtools sort - SRR2969245
rm SRR2969245.sam
samtools view -bS SRR2969246.sam | samtools sort - SRR2969246
rm SRR2969246.sam
samtools view -bS SRR2979473.sam | samtools sort - SRR2979473
rm SRR2979473.sam
samtools view -bS SRR2979475.sam | samtools sort - SRR2979475 
rm SRR2979475.sam 
samtools view -bS SRR2979476.sam | samtools sort - SRR2979476
rm SRR2979476.sam

Using Rsamtools for counting the reads aligned.

Creating files with the names of the bam files.

Some samples are single and other samples are pair-ended. We create the file BamFiles.txt with the following content.

SRR2969246.bam SRR2969245.bam SRR2969244.bam SRR2969243.bam SRR2969242.bam SRR2969241.bam SRR2969240.bam SRR2969239.bam SRR2969235.bam SRR2979476.bam SRR2969234.bam SRR2979475.bam SRR2969233.bam SRR2979473.bam SRR2969233.bam SRR2979473.bam SRR2969238.bam SRR2969237.bam SRR2969236.bam SRR2969232.bam SRR2969231.bam SRR2969230.bam

Executing R code

  library(Rsamtools)
  library(GenomicFeatures)
  library(GenomicAlignments)
  gtfFile = "Caenorhabditis_elegans/Ensembl/WBcel235/Annotation/Genes/genes.gtf" 
  txdb = makeTxDbFromGFF(gtfFile, format="gtf")
  genes = exonsBy(txdb, by="gene")
  dirActualData =  paste(getwd(),"/",sep="")
  sampleTableSingle = read.table("BamFiles.txt")
  fls = paste(dirActualData,sampleTableSingle[,1],sep="")
  bamLst = BamFileList(fls, index=character(),yieldSize=100000,obeyQname=TRUE)
  PRJNA297798 = summarizeOverlaps(features = genes,read=bamLst,
      mode="Union",
      singleEnd=TRUE,
      ignore.strand=TRUE,
      fragments=FALSE)
  metadatos = read.csv("PRJNA297798coldata.csv",header=TRUE,sep=",")
  metadatos[,"strain"] = factor(metadatos[,"strain"],levels=1:4,
               labels=c("daf-2(e1370);daf-16(mu86);Punc-119::gfp",
                        "daf-2(e1370);Punc-119::gfp",
                   "pmec-4::gfp","Punc-119::gfp"))
  metadatos[,"tissue"] = factor(metadatos[,"tissue"],levels=1:2,labels=c("Neurons","Whole worm"))
  metadatos[,"treatment"] = factor(metadatos[,"treatment"],levels=1:6,
               labels=c("daf-2(e1370); daf-16(mu86); Punc-119::gfp--sorted neurons",
                   "daf-2(e1370) neurons","N2; Pmec-4::GFP--sorted mechanosensory neurons",
                   "daf-2(e1370); Punc-119::gfp--sorted neurons","N2; Punc-119::gfp--sorted neurons",
                        "N2; Punc-119::gfp--unsorted/whole worm"))
  colData(PRJNA297798) = DataFrame(metadatos)
  colnames(PRJNA297798) = colData(PRJNA297798)[,"run"]
  save(PRJNA297798,file="PRJNA297798.rda")

We have a SummarizedExperiment::RangedSummarizedExperiment object.