TY - JOUR
T1 - Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing
AU - Gómez-Martín, Cristina
AU - Aparicio-Puerta, Ernesto
AU - van Eijndhoven, Monique A. J.
AU - Medina, José M.
AU - Hackenberg, Michael
AU - Pegtel, D. Michiel
N1 - Funding Information: This work was supported by Spanish Government ( AGL2017-88702-C2-2-R ) to M.H., Stitching Cancer Center Amsterdam ( CCA2021-9-77 ) to C.G.-M., and multiple grants awarded to D.M.P. including NWO Perspectief Cancer-ID, TKI-health Holland AQrate , Cancer Center Amsterdam Foundation , and Stichting MRD in Hodgkin Lymphoma. The authors would like to thank Dr. S. Gu for providing the TUT4/7 DKO HEK293T cells, and the usage of the computational infrastructure of the Computational Epigenomics Lab of the University of Granada. Funding Information: This work was supported by Spanish Government (AGL2017-88702-C2-2-R) to M.H. Stitching Cancer Center Amsterdam (CCA2021-9-77) to C.G.-M. and multiple grants awarded to D.M.P. including NWO Perspectief Cancer-ID, TKI-health Holland AQrate, Cancer Center Amsterdam Foundation, and Stichting MRD in Hodgkin Lymphoma. The authors would like to thank Dr. S. Gu for providing the TUT4/7 DKO HEK293T cells, and the usage of the computational infrastructure of the Computational Epigenomics Lab of the University of Granada. C.G.-M. performed the analysis together with M.A.J.v.E. E.A.-P. and J.M.M. M.A.J.v.E. performed the experiments. D.M.P. and M.H. co-designed and supervised this study. C.G.-M. and D.M.P. drafted the manuscript. All authors helped to edit the manuscript and final approval. D.M.P. and M.H. are co-founders of Exbiome BV of which D.M.P. acts as CSO. D.M.P. and M.H. are inventors on a related patent (PCT/EP2015/058614). Publisher Copyright: © 2023 The Author(s)
PY - 2023/5/22
Y1 - 2023/5/22
N2 - 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.
AB - 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.
KW - CP: Molecular biology
KW - isomiRs
KW - miRNAs
KW - randomized-end adapter protocols
UR - http://www.scopus.com/inward/record.url?scp=85159429934&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.crmeth.2023.100480
DO - https://doi.org/10.1016/j.crmeth.2023.100480
M3 - Article
C2 - 37323569
SN - 2667-2375
VL - 3
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 5
M1 - 100480
ER -