ShRNA-seq data analysis with edgeR.

Abstract

Pooled short hairpin RNA sequencing (shRNA-seq) screens are becoming increasinglypopular in functional genomics research, and there is a need to establishoptimal analysis tools to handle such data. Our open-source shRNA processingpipeline in edgeR provides a complete analysis solution for shRNA-seq screendata, that begins with the raw sequence reads and ends with a ranked lists ofcandidate shRNAs for downstream biological validation. We first summarize theraw data contained in a fastq file into a matrix of counts (samples in the columns,hairpins in the rows) with options for allowing mismatches and small shifts in hairpinposition. Diagnostic plots, normalization and differential representation analysiscan then be performed using established methods to prioritize results in a statisticallyrigorous way, with the choice of either the classic exact testing methodology or a generalized linear modelling that can handle complex experimental designs. Adetailed users' guide that demonstrates how to analyze screen data in edgeR alongwith a point-and-click implementation of this workflow in Galaxy are also provided.The edgeR package is freely available from http://www.bioconductor.org.