LiBiNorm: an htseq-count analogue with improved normalisation of Smart-seq2 data and library preparation diagnostics

Abstract

Protocols for preparing RNA sequencing (RNA-seq) libraries, most prominently “Smart-seq” variations, introduce global biases that can have a significant impact on the quantification of gene expression levels. This global bias can lead to drastic over- or under-representation of RNA in non-linear length-dependent fashion due to enzymatic reactions during cDNA production. It is currently not corrected by any RNA-seq software, which mostly focus on local bias in coverage along RNAs. This paper describes LiBiNorm, a simple command line program that mimics the popular htseq-count software and allows diagnostics, quantification, and global bias removal. LiBiNorm outputs gene expression data that has been normalized to correct for global bias introduced by the Smart-seq2 protocol. In addition, it produces data and several plots that allow insights into the experimental history underlying library preparation. The LiBiNorm package includes an R script that allows visualization of the main results. LiBiNorm is the first software application to correct for the global bias that is introduced by the Smart-seq2 protocol. It is freely downloadable at http://www2.warwick.ac.uk/fac/sci/lifesci/research/libinorm.