September 22, 2019  |  

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

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

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.

Journal: Nature biotechnology
DOI: 10.1038/nbt.2972
Year: 2014

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