Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic equimolar pool

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. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed sequencing bias and reproducibility using an equimolar pool of 1,152 small RNA sequences ranging from 15-90 nt, and primarily comprised of annotated human microRNAs. We observed extensive protocol-specific and sequence-specific bias that was largely mitigated in protocols employing sequencing adapters with randomized end-nucleotides. We find that sequencing bias is highly reproducible across labs using the same library preparation technologies, and use the data to calculate inter-protocol bias correction factors. 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. A single pool comprised of 1,152 synthetic RNAs was sequenced by 9 different labs, using 1 common library construction protocol (TruSeq), and at least one additional protocol of their choice. A total of 20 sample libraries was produced, each in quadruplicate.