학술논문
Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories
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article
Author
Giraldez, MD; Spengler, RM; Etheridge, A; Godoy, PM; Barczak, AJ; Srinivasan, S; De Hoff, PL; Tanriverdi, K; Courtright, A; Lu, S; Khoory, J; Rubio, R; Baxter, D; Driedonks, TAP; Buermans, HPJ; Nolte-‘t Hoen, ENM; Jiang, H; Wang, K; Ghiran, I; Wang, Y; Van Keuren-Jensen, K; Freedman, JE; Woodruff, PG; Laurent, LC; Erle, DJ; Galas, DJ; Tewari, M
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Abstract
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.