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

• Main Authors: Aparicio-Puerta, E. (Author), Gómez-Martín, C. (Author), Hackenberg, M. (Author), Medina, J.M (Author), Pegtel, D.M (Author), van Eijndhoven, M.A.J (Author)
• Format: Article
• Language: English
• Published: Cell Press 2023
• Subjects: CP: Molecular biology; isomiRs; miRNAs; randomized-end adapter protocols
• Online Access: View Fulltext in Publisher; View in Scopus
• ISBN: 26672375 (ISSN)
• DOI: 10.1016/j.crmeth.2023.100480

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. © 2023 The Author(s)