---+ 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) * BWA read mapper software * [[http://bio-bwa.sourceforge.net/][Download BWA]] * To build, just type "make" in the source code directory. * To install, move the executable "bwa" to somewhere in your $PATH, like $HOME/local/bin. * For usage see the [[http://bio-bwa.sourceforge.net/bwa.shtml][BWA manual]]. * [[http://samtools.sourceforge.net/][SAMtools]] * R statistics package * [[http://cran.r-project.org/][Download and install R]] * Bioconductor R modules * library(edgeR) * library(DESEQ) * library(Rsamtools) * HTSeq (Python package) * You may need to install python (package python27 if you use [[http://macports.org][MacPorts]]) * Install Numpy - (package py-numpy if you use [[http://macports.org][MacPorts]]) * [[http://www-huber.embl.de/users/anders/HTSeq/doc/install.html][Install HTSeq]] ---++ Commands ---+++ Align reads to reference genome First, index your genome so BWA can map read to it: %BR% <code>$bwa index REL606.fna</code> %BR% Then, align each data set: %BR% <code>$bwa aln REL606.fna dataset1.fastq > datasetX.sai </code> %BR% And convert to SAM format (assumes single-end data): <code>$bwa samse REL606.fna datasetX.sai datasetX.fastq > datasetX.sam </code> %BR% And convert to BAM format (assumes single-end data): %BR% <code>$samtools faidx REL606.fna </code> %BR% <code>$samtools import REL606.fna datasetX.sam datasetX.unsorted.bam </code> %BR% <code>$samtools sort datasetX.unsorted.bam datasetX </code> %BR% <code>$samtools index datasetX.bam </code> %BR% ---+++ Analyze differential gene expression library(DESEQ)
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Topic revision: r2 - 2012-01-28 - JeffreyBarrick