title: Normalization of RNA-sequencing data from samples with varying mRNA levels.
abstract: Methods for normalization of RNA-sequencing gene expression data commonly assume equal total expression between compared samples. In contrast, scenarios of global gene expression shifts are many and increasing. Here we compare the performance of three normalization methods when polyA(+) RNA content fluctuates significantly during zebrafish early developmental stages. As a benchmark we have used reverse transcription-quantitative PCR. The results show that reads per kilobase per million (RPKM) and trimmed mean of M-values (TMM) normalization systematically leads to biased gene expression estimates. Biological scaling normalization (BSN), designed to handle differences in total expression, showed improved accuracy compared to the two other methods in estimating transcript level dynamics. The results have implications for past and future studies using RNA-sequencing on samples with different levels of total or polyA(+) RNA.
authors.affiliation: BasAM, Norwegian School of Veterinary Science, Oslo, Norway.
authors.forename: Håvard
authors.initials: H
authors.lastname: Aanes
authors.affiliation: Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore, Singapore.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Winata
authors.affiliation: BasAM, Norwegian School of Veterinary Science, Oslo, Norway.
authors.forename: Lars F
authors.initials: LF
authors.lastname: Moen
authors.affiliation: Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, and Norwegian Center for Stem Cell Research, Oslo, Norway.
authors.forename: Olga
authors.initials: O
authors.lastname: Østrup
authors.affiliation: Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore, Singapore.
authors.forename: Sinnakaruppan
authors.initials: S
authors.lastname: Mathavan
authors.affiliation: Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, and Norwegian Center for Stem Cell Research, Oslo, Norway.
authors.forename: Philippe
authors.initials: P
authors.lastname: Collas
authors.affiliation: Department of Informatics, University of Oslo, Oslo, Norway.
authors.forename: Torbjørn
authors.initials: T
authors.lastname: Rognes
authors.affiliation: BasAM, Norwegian School of Veterinary Science, Oslo, Norway.
authors.forename: Peter
authors.initials: P
authors.lastname: Aleström
mesh_terms: D000818:Animals; D001483:Base Sequence / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D015027:Zebrafish / Q000235:genetics
chemical_list: D012333:RNA, Messenger