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Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories

  • Author(s): 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
  • et al.

Published Web Location

https://doi.org/10.1101/113050
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.

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