IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution

Monique A J van Eijndhoven; Chantal Scheepbouwer; Ernesto Aparicio-Puerta; Michael Hackenberg; D Michiel Pegtel

doi:10.1016/j.xpro.2023.102645

IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution

STAR Protoc. 2023 Dec 15;4(4):102645. doi: 10.1016/j.xpro.2023.102645. Epub 2023 Oct 19.

Authors

Monique A J van Eijndhoven¹, Chantal Scheepbouwer², Ernesto Aparicio-Puerta³, Michael Hackenberg³, D Michiel Pegtel⁴

Affiliations

¹ Amsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Pathology, 1081 HV Amsterdam, the Netherlands; Cancer Center Amsterdam, Imaging and Biomarkers, 1081 HV Amsterdam, the Netherlands. Electronic address: m.vaneijndhoven@amsterdamumc.nl.
² Amsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Pathology, 1081 HV Amsterdam, the Netherlands; Amsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Neurosurgery, 1081 HV Amsterdam, the Netherlands; Cancer Center Amsterdam, Cancer Biology, 1081 HV Amsterdam, the Netherlands.
³ Department of Genetics, Faculty of Science, University of Granada, 18071 Granada, Spain; Bioinformatics Laboratory, Biotechnology Institute, Centro de Investigacion Biomedica, PTS, Avda. del Conocimiento s/n, 18100 Granada, Spain; Instituto de Investigacion Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain; Excellence Research Unit ''Modelling Nature'' (MNat), University of Granada, 18071 Granada, Spain.
⁴ Amsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Pathology, 1081 HV Amsterdam, the Netherlands; Cancer Center Amsterdam, Imaging and Biomarkers, 1081 HV Amsterdam, the Netherlands. Electronic address: d.pegtel@amsterdamumc.nl.

Abstract

Besides canonical microRNAs (miRNAs), sequence-based variations called isomiRs have biological relevance and diagnostic potential; however, accurate calling of these post-transcriptional modifications is challenging, especially for low input samples. Here, we present IsoSeek, a sequencing protocol that reduces ligation and PCR amplification bias and improves the accuracy of miRNA detection in low input samples. We describe steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, and sequencing, followed by detailed procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to C. Gómez-Martín et al. (2023)¹ and Van Eijndhoven et al. (2021).².

Keywords: Bioinformatics; Biotechnology and Bioengineering; Genetics; Genomics.

MeSH terms

Gene Library
High-Throughput Nucleotide Sequencing / methods
MicroRNAs* / genetics
Nucleotides / genetics
Sequence Analysis, RNA / methods

Substances

MicroRNAs
Nucleotides