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

Cristina Gómez-Martín; Ernesto Aparicio-Puerta; Monique A J van Eijndhoven; José M Medina; Michael Hackenberg; D Michiel Pegtel

doi:10.1016/j.crmeth.2023.100480

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

Cell Rep Methods. 2023 May 16;3(5):100480. doi: 10.1016/j.crmeth.2023.100480. eCollection 2023 May 22.

Authors

Cristina Gómez-Martín^{1

2}, Ernesto Aparicio-Puerta³, Monique A J van Eijndhoven^{1

2}, José M Medina^{4

5}, Michael Hackenberg^{4

5

6

7}, D Michiel Pegtel^{1

2}

Affiliations

¹ Amsterdam UMC Location Vrije Universiteit Amsterdam, Pathology, De Boelelaan, 1117 Amsterdam, the Netherlands.
² Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, the Netherlands.
³ Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany.
⁴ Department of Genetics, Faculty of Science, University of Granada, 18071 Granada, Spain.
⁵ Bioinformatics Laboratory, Biotechnology Institute, Centro de Investigación Biomédica, PTS, Avda. del Conocimiento s/n, 18100 Granada, Spain.
⁶ Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain.
⁷ Excellence Research Unit "Modelling Nature" (MNat), University of Granada, 18071 Granada, Spain.

Abstract

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.

Keywords: isomiRs; miRNAs; randomized-end adapter protocols.

MeSH terms

Base Sequence
HEK293 Cells
Humans
MicroRNAs* / genetics
Sequence Analysis, RNA

Substances

MicroRNAs