title: Detecting and correcting systematic variation in large-scale RNA sequencing data.
abstract: High-throughput RNA sequencing (RNA-seq) enables comprehensive scans of entire transcriptomes, but best practices for analyzing RNA-seq data have not been fully defined, particularly for data collected with multiple sequencing platforms or at multiple sites. Here we used standardized RNA samples with built-in controls to examine sources of error in large-scale RNA-seq studies and their impact on the detection of differentially expressed genes (DEGs). Analysis of variations in guanine-cytosine content, gene coverage, sequencing error rate and insert size allowed identification of decreased reproducibility across sites. Moreover, commonly used methods for normalization (cqn, EDASeq, RUV2, sva, PEER) varied in their ability to remove these systematic biases, depending on sample complexity and initial data quality. Normalization methods that combine data from genes across sites are strongly recommended to identify and remove site-specific effects and can substantially improve RNA-seq studies.
authors.affiliation: 1] Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA. [2] The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, New York, New York, USA. [3].
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.affiliation: 1] Chair of Bioinformatics Research Group, Boku University Vienna, Vienna, Austria. [2].
authors.forename: Paweł P
authors.initials: PP
authors.lastname: Łabaj
authors.affiliation: 1] Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA. [2] The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, New York, New York, USA. [3].
authors.forename: Paul
authors.initials: P
authors.lastname: Zumbo
authors.affiliation: Chair of Bioinformatics Research Group, Boku University Vienna, Vienna, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Sykacek
authors.affiliation: Department of Bioinformatics, WEHI, Melbourne, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, Schools of Life Sciences and Pharmacy, Fudan University, Shanghai, China.
authors.forename: Leming
authors.initials: L
authors.lastname: Shi
authors.affiliation: School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
authors.forename: John
authors.initials: J
authors.lastname: Phan
authors.affiliation: School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
authors.forename: Po-Yen
authors.initials: PY
authors.lastname: Wu
authors.affiliation: School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
authors.forename: May
authors.identifier: 0000000339613608
authors.initials: M
authors.lastname: Wang
authors.affiliation: Center for Genomics and Division of Microbiology &Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, California, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Wang
authors.affiliation: National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Thierry-Mieg
authors.affiliation: National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Thierry-Mieg
authors.affiliation: 1] Chair of Bioinformatics Research Group, Boku University Vienna, Vienna, Austria. [2] University of Warwick, Coventry, UK.
authors.forename: David P
authors.initials: DP
authors.lastname: Kreil
authors.affiliation: 1] Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA. [2] The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, New York, New York, USA. [3] The Feil Family Brain and Mind Research Institute, New York, New York, USA.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Mason
mesh_terms: D011786:Quality Control; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods*