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.