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
    • 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
  • Download and install R
• Bioconductor R modules
  • library(edgeR)
  • library(DESEQ)

Commands

Align reads to reference genome

Using BWA

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)