PRJNA266927

Guillermo Ayala

2024-04-02

Download the fastq files from ENA

wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/005/SRR1656855/SRR1656855.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/006/SRR1656856/SRR1656856.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/007/SRR1656857/SRR1656857.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/008/SRR1656858/SRR1656858.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/009/SRR1656859/SRR1656859.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/000/SRR1656860/SRR1656860.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/001/SRR1656861/SRR1656861.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/002/SRR1656862/SRR1656862.fastq.gz

Download the index file for bowtie and bowtie2

wget ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Drosophila_melanogaster/Ensembl/BDGP6/Drosophila_melanogaster_Ensembl_BDGP6.tar.gz

Generating the directories with the index files

gzip -d Drosophila_melanogaster_Ensembl_BDGP6.tar.gz
tar xvf Drosophila_melanogaster_Ensembl_BDGP6.tar

Short read alignment using bowtie2

bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656855.fastq.gz -S  SRR1656855.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656856.fastq.gz -S  SRR1656856.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656857.fastq.gz -S  SRR1656857.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656858.fastq.gz -S  SRR1656858.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656859.fastq.gz -S  SRR1656859.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656860.fastq.gz -S  SRR1656860.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656861.fastq.gz -S  SRR1656861.sam
bowtie2 -x Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome -U SRR1656862.fastq.gz -S  SRR1656862.sam

From sam to bam using samtools and sorting the reads

samtools view -bS SRR1656855.sam | samtools sort - SRR1656855
rm SRR1656855.sam
samtools view -bS SRR1656856.sam | samtools sort - SRR1656856
rm SRR1656856.sam
samtools view -bS SRR1656857.sam | samtools sort - SRR1656857
rm SRR1656857.sam
samtools view -bS SRR1656858.sam | samtools sort - SRR1656858
rm SRR1656858.sam
samtools view -bS SRR1656859.sam | samtools sort - SRR1656859
rm SRR1656859.sam
samtools view -bS SRR1656860.sam | samtools sort - SRR1656860
rm SRR1656860.sam
samtools view -bS SRR1656861.sam | samtools sort - SRR1656861
rm SRR1656861.sam
samtools view -bS SRR1656862.sam | samtools sort - SRR1656862
rm SRR1656862.sam

Creating files with the names of the bam files.

SRR1656855.bam
SRR1656856.bam
SRR1656857.bam
SRR1656858.bam
SRR1656859.bam
SRR1656860.bam
SRR1656861.bam
SRR1656862.bam

Using Rsamtools for counting the reads aligned.

library(Rsamtools)
library(GenomicFeatures)
library(GenomicAlignments)
gtfFile = "Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf" 
txdb = makeTxDbFromGFF(gtfFile, format="gtf")
genes = exonsBy(txdb, by="gene")
dirActualData =  paste(getwd(),"/",sep="")
sampleTableSingle = read.table("BamSingle.txt")
fls = paste(dirActualData,sampleTableSingle[,1],sep="")
bamLst = BamFileList(fls, index=character(),yieldSize=100000,obeyQname=TRUE)
PRJNA266927 = summarizeOverlaps(features = genes,read=bamLst,
   mode="Union",
      singleEnd=TRUE,
      ignore.strand=TRUE,
      fragments=FALSE)
Run = c("SRR1656855","SRR1656856","SRR1656857","SRR1656858","SRR1656859","SRR1656860",
        "SRR1656861","SRR1656862")
Treatment = c(1,1,2,2,3,3,4,4)
Treatment = factor(Treatment,levels=1:4,labels=c("NC","NOTCH","ESG","TUMOR"))
colData(PRJNA266927) = DataFrame(Treatment)
save(PRJNA266927,file="PRJNA266927.rda")

Short read alignment using samtools

What else can we obtain using samtools?

tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656855_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656855.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656856_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656856.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656857_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656857.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656858_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656858.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656859_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656859.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656860_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656860.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656861_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656861.fastq.gz
tophat -p 8 -G Drosophila_melanogaster/Ensembl/BDGP6/Annotation/Genes/genes.gtf -o SRR1656862_thout Drosophila_melanogaster/Ensembl/BDGP6/Sequence/Bowtie2Index/genome SRR1656862.fastq.gz