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

  • Author(s): Giraldez, Maria D
  • Spengler, Ryan M
  • Etheridge, Alton
  • Godoy, Paula M
  • Barczak, Andrea J
  • Srinivasan, Srimeenakshi
  • De Hoff, Peter L
  • Tanriverdi, Kahraman
  • Courtright, Amanda
  • Lu, Shulin
  • Khoory, Joseph
  • Rubio, Renee
  • Baxter, David
  • Driedonks, Tom AP
  • Buermans, Henk PJ
  • Nolte-'t Hoen, Esther NM
  • Jiang, Hui
  • Wang, Kai
  • Ghiran, Ionita
  • Wang, Yaoyu E
  • Van Keuren-Jensen, Kendall
  • Freedman, Jane E
  • Woodruff, Prescott G
  • Laurent, Louise C
  • Erle, David J
  • Galas, David J
  • Tewari, Muneesh
  • et al.

Published Web Location

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.

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