Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing

IsomiRs, sequence variants of mature microRNAs, are usually detected and quantified using high-throughput sequencing. Many examples of their biological relevance have been reported, but sequencing artifacts identified as artificial variants might bias biological inference and therefore need to be ideally avoided. We conducted a comprehensive evaluation of 10 different small RNA sequencing protocols, exploring both a theoretically isomiR-free pool of synthetic miRNAs and HEK293T cells. We calculated that, with the exception of two protocols, less than 5% of miRNA reads can be attributed to library preparation artifacts. Randomized-end adapter protocols showed superior accuracy, with 40% of true biological isomiRs. Nevertheless, we demonstrate concordance across protocols for selected miRNAs in non-templated uridyl additions. Notably, NTA-U calling and isomiR target prediction can be inaccurate when using protocols with poor single-nucleotide resolution. Our results highlight the relevance of protocol choice for biological isomiRs detection and annotation, which has key potential implications for biomedical applications. [Display omitted] •IsomiRs are not a consequence of library artifacts in most RNA-seq protocols•Randomized adapter protocols outperform fixed adapters in isomiR calling accuracy•NTA-U calling is dependent on high-single-nucleotide-resolution performance•NTA-U isomiR calling affects mRNA target prediction Sequence variants (isomiRs) of mature microRNAs can be detected by high-throughput sequencing methods. However, because isomiRs differ from the canonical (mature) miRNA sequence often by only a single nucleotide, most protocols introduce errors that may negatively influence biological interpretation. To investigate how sequencing protocols impact isomiR calling, we conducted a comprehensive comparison of 10 small RNA-seq protocols, providing a comprehensive reference guide for miRNA/isomiR interpretation by sequencing. Gómez-Martín et al. demonstrate that isomiRs are non-artifactual variants of miRNAs and that most small RNA sequencing protocols, especially those with randomized adapters, detect them with high accuracy. Single-nucleotide accuracy is crucial for making predictions about biological function. The work provides a reference for researchers interested in small ncRNA.