rnaSeqPipelineGLBRC.py

Purpose:

Implementation of the Gasch lab RNA-Seq pipeline.

Input :

A text file with RNA-Seq fastq files to be processed.

Please use a dedicated directory for running pipeline. Create your directory and copy your fastq files into that directory. Generate input file by moving into working directory and running /bin/ls *.fastq > input.txt

Required Parameters:

-f input.txt

To run default enter:  /home/GLBRCORG/mplace/scripts/rnaSeqPipelineGLBRC.py -f input.txt

Optional Parameters:

-r  this will use "-s reverse" parameter for HTSeq.

-a  bwamem changes aligner from default bowtie2 to bwa mem

-ref  change default reference, usage:  -ref y22-3
    
	Current Reference List:

	R64-1-1 -- default equivalent to UCSC sacCer3

	Y22-3   -- GLBRC assembly 

Outline of steps & commands used in pipeline:

1. Trimmomatic

/opt/bifxapps/Trimmomatic-0.30/trimmomatic-0.30.jar SE -phred33 input_fastq 
outFile LEADING:3 TRAILING:3 SLIDINGWINDOW:3:30 MINLEN:36

2. Fastqc

/opt/bifxapps/FastQC/fastqc trimmed_fastq

3. Alignment

Bowtie2 -p 8 --phred33 -N 1 -x  referenceFile -U  trimmed_fastq -S outFile

or

bwa mem -t 8 -M referenceFile trimmed_fastq

4. Picard-tools

/opt/bifxapps/picard-tools/CleanSam.jar I= samFile O=outFile

/opt/bifxapps/picard-tools/AddOrReplaceReadGroups.jar 'I=', inFile 

O=outFile SO=coordinate LB=S288C_reference_sequence_R64-1-1_20110203.fasta

PL=ILLUMINA PU=unk RGSM=SampleName

5. samtools

sam to bam: samtools view -bS -t reference.fsa.fai -o bam samFile

  sort bam: samtools sort input_bam sorted_Bam 

 index bam: samtools index sorted_Bam

6. HTSeq

/opt/bifxapps/python/bin/htseq-count -t CDS -i Parent inputFile Ref_gff

 -- if '-r' specfied -s reverse will be used as well

7. RPKM

/home/GLBRCORG/mplace/bin/Normalize.jar dir Ref_gff RPKM.results --gene

8. bam2wig.pl

/opt/bifxapps/biotoolbox/scripts/bam2wig.pl --in bamFile --pos mid --strand --rpm --out outFile

9. findreplace_WIG.pl

/home/GLBRCORG/mplace/scripts/findreplace_WIG.pl

The results are organized into the following subdirectories:

alignments/ 

fastq/

fastqc/

htseq/ 

log/ 

wig/ 

RPKM results are written to a file called: RPKM.results

This script is designed to run on the GLBRC scarcity servers 6-10.