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Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5’ and 3’ ends of small RNAs. Studies have shown that adapter ligation introduces a significant but widely unappreciated bias in the results of high-throughput small RNA sequencing. We show that due to this bias the two widely used Illumina library preparation protocols produce strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that are >5-fold differentially detected and some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. In an attempt to mitigate this bias, the new Bioo Scientific NEXTflex V2 protocol utilizes a pool of adapters with random nucleotides at the ligation boundary. We show that this protocol is able to detect robustly several miRNAs that evade capture by the Illumina-based methods. While these analyses do not indicate a definitive gold standard for small RNA library preparation, the results of the NEXTflex protocol do correlate best with RT-qPCR. As increasingly more laboratories seek to study small RNAs, researchers should be aware of the extent to which the results may differ with different protocols, and should make an informed decision about the protocol that best fits their study.

Next-generation deep sequencing of small RNAs has unveiled the complexity of the microRNA (miRNA) transcriptome, which is in large part due to the diversity of miRNA sequence variants ("isomiRs"). Changes to a miRNA's seed sequence (nucleotides 2-8), including shifted start positions, can redirect targeting to a dramatically different set of RNAs and alter biological function. We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells (widely used as a surrogate for the study of pancreatic beta cells) and developed a bioinformatic analysis pipeline to profile isomiR diversity. Additionally, we applied the pipeline to recently published small RNA-seq data from primary human beta cells and whole islets and compared the miRNA profiles with that of MIN6. We found that: (1) the miRNA expression profile in MIN6 cells is highly correlated with those of primary human beta cells and whole islets; (2) miRNA loci can generate multiple highly expressed isomiRs with different 5'-start positions (5'-isomiRs); (3) isomiRs with shifted start positions (5'-shifted isomiRs) are highly expressed, and can be as abundant as their unshifted counterparts (5'-reference miRNAs). Finally, we identified 10 beta cell miRNA families as candidate regulatory hubs in a type 2 diabetes (T2D) gene network. The most significant candidate hub was miR-29, which we demonstrated regulates the mRNA levels of several genes critical to beta cell function and implicated in T2D. Three of the candidate miRNA hubs were novel 5'-shifted isomiRs: miR-375+1, miR-375-1 and miR-183-5p+1. We showed by in silico target prediction and in vitro transfection studies that both miR-375+1 and miR-375-1 are likely to target an overlapping, but distinct suite of beta cell genes compared to canonical miR-375. In summary, this study characterizes the isomiR profile in beta cells for the first time, and also highlights the potential functional relevance of 5'-shifted isomiRs to T2D.