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Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories

By MD Giraldez, RM Spengler, A Etheridge, PM Godoy, AJ Barczak, S Srinivasan, PL De Hoff, K Tanriverdi, A Courtright, S Lu, J Khoory, R Rubio, D Baxter, TAP Driedonks, HPJ Buermans, ENM Nolte-‘t Hoen, H. Jiang, K Wang, I Ghiran, Y. Wang, K Van Keuren-Jensen, JE Freedman, PG Woodruff, LC Laurent, DJ Erle, DJ Galas, M Tewari

Posted 17 May 2017
bioRxiv DOI: 10.1101/113050

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

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