PT  - JOURNAL ARTICLE
AU  - MD Giraldez
AU  - RM Spengler
AU  - A Etheridge
AU  - PM Godoy
AU  - AJ Barczak
AU  - S Srinivasan
AU  - PL De Hoff
AU  - K Tanriverdi
AU  - A Courtright
AU  - S Lu
AU  - J Khoory
AU  - R Rubio
AU  - D Baxter
AU  - TAP Driedonks
AU  - HPJ Buermans
AU  - ENM Nolte-‘t Hoen
AU  - H Jiang
AU  - K Wang
AU  - I Ghiran
AU  - Y Wang
AU  - K Van Keuren-Jensen
AU  - JE Freedman
AU  - PG Woodruff
AU  - LC Laurent
AU  - DJ Erle
AU  - DJ Galas
AU  - M Tewari
TI  - Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories
AID  - 10.1101/113050
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 113050
4099  - http://biorxiv.org/content/early/2017/05/17/113050.short
4100  - http://biorxiv.org/content/early/2017/05/17/113050.full
AB  - 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.