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.
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We greatly appreciate the contribution and distribution of reference sample RNA from L. Shi (FDA) and his valuable interactions to assist in the planning of this study. This work was supported with funding from the National Institutes of Health (NIH), including R01HG006798, R01NS076465, R24RR032341, as well as funds from the Irma T. Hirschl and Monique Weill-Caulier Charitable Trusts and the STARR Consortium (I7-A765).
We thank the following contributors for their technical wisdom, including laboratory expertise, data analysis and bioinformatics contributions, and technical design guidance and consultation. Without their help, this study would not have been possible: D. Stopka (Memorial Sloan-Kettering Cancer Institute), G. Grove (Penn State Univ.), D. Hannon (Penn State Univ.), K. Jones (NIH/NCI/SAIC), C. Raley (NIH/NCI/SAIC), H. O'Geen (UC Davis), D. Zheng (Univ. Illinois-Urbana), O. Nguyen (UC Davis), Z.-W. Lu (UC Davis), J. Spisak (Cornell Univ.), D. Lin (NIH/NIAID), J. Pillardy (Cornell Univ.), P.-Y. Wu (Georgia Institute of Technology), J. Phan (Emory Univ.), D. Oschwald (New York Genome Center), H. Arnold (PerkinElmer), S. Tyndale (Univ. Southern California), H. Truong (Univ. Southern California), Y. Zhang (Univ. Florida), N. Panayotova (Univ. Florida), D. Moraga (Univ. Florida), S. Shanker (Univ. Florida), and N. Barker (US Army Environmental Quality Research Program).
We would also like to thank the platform vendors, Illumina, Life Technologies, Pacific Biosciences and Roche Life Sciences, for their support of this study, and their distinguished scientists for providing technical expertise and assistance in study designs, protocols, new methods development and significant contributions of reagents and sequencing kits. In particular, alphabetically by vendor: G. Schroth (Illumina); M. Gallad, J. Smith, T. Bittick, R. Setterquist and G. Scott (Life Technologies); J. Korlach, S. Turner and E. Tseng (Pacific Biosciences); and K. Fredrickson and C. Teiling (Roche Life Sciences).
We are sincerely appreciative of the Association of Biomolecular Resource Facilities (ABRF) for supporting this study and the contributing ABRF Research Groups. Special thanks to our ABRF executive board liaison A. Perera (Stowers Institute for Medical Research).
The authors declare no competing financial interests.
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Li, S., Tighe, S., Nicolet, C. et al. Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study. Nat Biotechnol 32, 915–925 (2014). https://doi.org/10.1038/nbt.2972
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