Analyzing RNA-Seq data for differential gene expression

Materials and Software

  • Data files
    • RNA-Seq data in FASTQ format (Ex: dataset1.fastq, dataset2.fastq)
  • Genome sequence files
    • Genome sequence in FASTA format (Ex: REL606.fna)
    • Genome sequence gene annotations in GFF3 format (Ex: REL606.gff3)
  • Read mapper software
    • BWA
      • Download BWA
      • To build, just type "make" in the source code directory.
      • To install, move the executable "bwa" to somewhere in your $PATH, like $HOME/local/bin.
      • For usage see the BWA manual.
    • Bowtie2
      • [[http://bowtie-bio.sourceforge.net][Download Bowtie[[
      • To build, just type "make" in the source code directory.
      • Add this directory to your $PATH or move bowtie, and bowtie-* star executable to your $PATH
  • breseq
  • R statistics package
  • Bioconductor R modules
    • library(edgeR)
    • library(DESEQ)

Commands

Align reads to reference genome

Using BWA

First, index your genome so BWA can map read to it:
$bwa index REL606.fna

Then, align each data set:
$bwa aln REL606.fna dataset1.fastq > datasetX.sai

And convert to SAM format (assumes single-end data): $bwa samse REL606.fna datasetX.sai datasetX.fastq > datasetX.sam

Using bowtie2

First, index your genome so bowtie2 can map read to it:
$bowtie2-build REL606.fna REL606

Then, align each data set:
$bowtie2 -x REL606 -U datasetX.fastq --phred33 -S REL606.sam

Optionally, add the --local flag if your reads do not map end-to-end.

Count reads mapping to genes

breseq RNASEQ -f REL606.fna -r REL606.gbk -o datasetX.count.tab datasetX.sam

Convert alignments to BAM

And convert to BAM format (assumes single-end data):
$samtools faidx REL606.fna
$samtools import REL606.fna datasetX.sam datasetX.unsorted.bam
$samtools sort datasetX.unsorted.bam datasetX
$samtools index datasetX.bam

Now you can use IGV to view them.

Analyze differential gene expression

library(DESEQ)

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 Barrick Lab  >  ProtocolList  >  ProtocolsRNASeqDifferentialExpression

Topic revision: r3 - 30 Jan 2012 - 06:45:56 - Main.JeffreyBarrick
 
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