- 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.
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.