title: Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories.
abstract: RNA sequencing is an increasingly popular technology for genome-wide analysis of transcript sequence and abundance. However, understanding of the sources of technical and interlaboratory variation is still limited. To address this, the GEUVADIS consortium sequenced mRNAs and small RNAs of lymphoblastoid cell lines of 465 individuals in seven sequencing centers, with a large number of replicates. The variation between laboratories appeared to be considerably smaller than the already limited biological variation. Laboratory effects were mainly seen in differences in insert size and GC content and could be adequately corrected for. In small-RNA sequencing, the microRNA (miRNA) content differed widely between samples owing to competitive sequencing of rRNA fragments. This did not affect relative quantification of miRNAs. We conclude that distributing RNA sequencing among different laboratories is feasible, given proper standardization and randomization procedures. We provide a set of quality measures and guidelines for assessing technical biases in RNA-seq data.
authors.affiliation: 1] Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. [2] Netherlands Bioinformatics Centre, Leiden, The Netherlands.
authors.forename: Peter A C
authors.initials: PA
authors.lastname: 't Hoen
authors.forename: Marc R
authors.initials: MR
authors.lastname: Friedländer
authors.forename: Jonas
authors.initials: J
authors.lastname: Almlöf
authors.forename: Michael
authors.initials: M
authors.lastname: Sammeth
authors.forename: Irina
authors.initials: I
authors.lastname: Pulyakhina
authors.forename: Seyed Yahya
authors.initials: SY
authors.lastname: Anvar
authors.forename: Jeroen F J
authors.initials: JF
authors.lastname: Laros
authors.forename: Henk P J
authors.initials: HP
authors.lastname: Buermans
authors.forename: Olof
authors.initials: O
authors.lastname: Karlberg
authors.forename: Mathias
authors.initials: M
authors.lastname: Brännvall
authors.forename: Johan T
authors.initials: JT
authors.lastname: den Dunnen
authors.forename: Gert-Jan B
authors.initials: GJ
authors.lastname: van Ommen
authors.forename: Ivo G
authors.initials: IG
authors.lastname: Gut
authors.forename: Roderic
authors.initials: R
authors.lastname: Guigó
authors.forename: Xavier
authors.initials: X
authors.lastname: Estivill
authors.forename: Ann-Christine
authors.initials: AC
authors.lastname: Syvänen
authors.forename: Emmanouil T
authors.initials: ET
authors.lastname: Dermitzakis
authors.forename: Tuuli
authors.initials: T
authors.lastname: Lappalainen
mesh_terms: D005260:Female; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods / Q000592:standards*
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger
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