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Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories
Maria D. Giraldez,
Ryan M. Spengler,
Paula Maria Godoy,
Andrea J. Barczak,
Peter L. De Hoff,
Tom A. P. Driedonks,
Hank P. J. Buermans,
Esther N. M. Nolte-‘t Hoen,
Kendall Van Keuren-Jensen,
Jane E. Freedman,
Prescott G. Woodruff,
Louise C. Laurent,
David J Erle,
David J. Galas,
Posted 17 May 2017
bioRxiv DOI: 10.1101/113050
Posted 17 May 2017
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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