Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.
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.
Institute for Systems Biology
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 A P; Buermans, Henk P J; Nolte-'t Hoen, Esther N M; 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; and Tewari, Muneesh, "Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling." (2018). Articles, Abstracts, and Reports. 548.