Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

Sheng Li, Scott W. Tighe, Charles M. Nicolet, Deborah Grove, Shawn Levy, William Farmerie, Agnes Viale, Chris Wright, Peter A. Schweitzer, Yuan Gao, Dewey Kim, Joe Boland, Belynda Hicks, Ryan Kim, Sagar Chhangawala, Nadereh Jafari, Nalini Raghavachari, Jorge Gandara, Natàlia Garcia-Reyero, Cynthia HendricksonDavid Roberson, Jeffrey A. Rosenfeld, Todd Smith, Jason G. Underwood, May Wang, Paul Zumbo, Don A. Baldwin, George S. Grills, Christopher E. Mason*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

118 Scopus citations

Abstract

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

Original languageEnglish (US)
Pages (from-to)915-925
Number of pages11
JournalNature biotechnology
Volume32
Issue number9
DOIs
StatePublished - Sep 1 2014

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

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