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Related Experiment Videos

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

Sheng Li1,2, Scott W Tighe3, Charles M Nicolet4

  • 1Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.

Nature Biotechnology
|August 25, 2014
PubMed

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Summary

This study compared RNA sequencing (RNA-seq) protocols and platforms. Results show high concordance for gene expression but variable splice junction and variant detection, aiding RNA-seq standardization.

Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • High-throughput RNA sequencing (RNA-seq) offers vast genomics discovery potential.
  • Diverse platforms and protocols necessitate reference data for standardization.
  • The Association of Biomolecular Resource Facilities (ABRF) initiated a study to address this need.

Purpose of the Study:

  • To evaluate and compare RNA sequencing performance across multiple platforms and protocols.
  • To establish a foundation for cross-platform standardization and evaluation of RNA-seq.
  • To assess the impact of different RNA preparation methods on sequencing outcomes.

Main Methods:

  • Conducted replicate RNA sequencing experiments across 15 laboratory sites.
  • Utilized reference RNA standards to test four protocols: poly-A selection, ribo-depletion, size selection, and degraded RNA.

Related Experiment Videos

  • Employed five sequencing platforms: Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS, and Roche 454.

    • Main Results:

    • Demonstrated high intra- and inter-platform concordance (Spearman rank R > 0.83) for gene expression measures on deep-count platforms.
    • Observed highly variable efficiency and cost for splice junction and variant detection across all tested platforms.
    • Found similar gene expression profiles for intact RNA using rRNA-depletion and poly-A enrichment.
    • Confirmed that rRNA depletion effectively analyzes degraded RNA samples.

    Conclusions:

    • RNA sequencing platforms exhibit high concordance for gene expression analysis.
    • Significant variability exists in splice junction and variant detection capabilities across platforms.
    • rRNA depletion is a robust method for analyzing both intact and degraded RNA samples.
    • The study provides essential data for the standardization and improvement of RNA sequencing technologies.