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

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 len...

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Bibliographic Details
Published in:PeerJ (San Francisco, CA) Vol. 7; p. e6222
Main Authors: Dyer, Nigel P., Shahrezaei, Vahid, Hebenstreit, Daniel
Format: Journal Article
Language:English
Published: United States PeerJ. Ltd 04.02.2019
PeerJ, Inc
PeerJ Inc
Subjects:
Bias
Biochemistry
Bioinformatics
Biology
Computational Biology
Computational Science
Computer programs
Data processing
Deoxyribonucleic acid
DNA
Enzymes
Gene expression
Genes
Genetic research
Genomes
Genomics
Global bias
Methods
Normalization
Ribonucleic acid
RNA
RNA sequencing
RNA-seq
Scientific software
Smart-seq2
Software
Technology application
Visualization (Computer)
United Kingdom > UK
Normalization
Smart-seq2
RNA-seq
Gene expression
Global bias
ISSN:2167-8359, 2167-8359
Online Access:Get full text
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Summary: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 .
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ISSN:2167-8359
2167-8359
DOI:10.7717/peerj.6222