Systematic assessment of next-generation sequencing for quantitative small RNA profiling: A-to-I editing pools. Systematic assessment of next-generation sequencing for quantitative small RNA profiling: A-to-I editing pools

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 reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies. Overall design: Six synthetic RNA pools were generated to assess the performance of three small RNA library preparation protocols: TruSeq, NEBNext and an in-house 4N_B protocol. The pools each contained a common set of 277 synthetic miRNA oligos in equimolar amounts, and oligos for 10 miRNAs with known miRNA Adenosine-to-Inosine (A-to-I) editing events. The six pools contained the edited (I) and unedited (A) variants of the oligos, keeping the total concentration of the miRNA the same (edited + unedited variant), but varying the percent edited oligo. The resulting six pools represented six different editing levels: 0%, 0.1%, 1%, 5%, 50% and 100% editing. Three labs sequenced each of the six pools in triplicate, using all three of the library preparation protocols.