Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

Nat Biotechnol. 2018 Sep;36(8):746-757. doi: 10.1038/nbt.4183. Epub 2018 Jul 16.


RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Publication types

  • Multicenter Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine / genetics
  • Humans
  • Inosine / genetics
  • MicroRNAs / blood
  • MicroRNAs / genetics*
  • MicroRNAs / standards
  • RNA Editing
  • Reference Standards
  • Reproducibility of Results
  • Sequence Analysis, RNA / methods*


  • MicroRNAs
  • Inosine
  • Adenosine