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

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. This SuperSeries is composed of the SubSeries listed below. Nine labs prepared small RNA-seq libraries from 4 common RNA pools: a) 1 equimolar pool of 1,152 synthetic RNAs, b) 2 pools of synthetic RNAs at differing relative concentrations in pool A and B, and c) human plasma RNA pooled from 11 healthy individuals. Each lab prepared libraries from the samples in quadruplicate, using a standardized TruSeq protocol, as well as at least one other protocol of their choice. Refer to individual Series for details.