IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution
Summary: 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 p...
| Published in: | STAR Protocols |
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| Main Authors: | , , , , |
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-12-01
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| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723006123 |
| _version_ | 1850078676312391680 |
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| author | Monique A.J. van Eijndhoven Chantal Scheepbouwer Ernesto Aparicio-Puerta Michael Hackenberg D. Michiel Pegtel |
| author_facet | Monique A.J. van Eijndhoven Chantal Scheepbouwer Ernesto Aparicio-Puerta Michael Hackenberg D. Michiel Pegtel |
| author_sort | Monique A.J. van Eijndhoven |
| collection | DOAJ |
| container_title | STAR Protocols |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-f333a2f9af6b43af94de6e9a12fc66dd |
| institution | Directory of Open Access Journals |
| issn | 2666-1667 |
| language | English |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| spelling | doaj-art-f333a2f9af6b43af94de6e9a12fc66dd2025-08-20T00:14:15ZengElsevierSTAR Protocols2666-16672023-12-014410264510.1016/j.xpro.2023.102645IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolutionMonique A.J. van Eijndhoven0Chantal Scheepbouwer1Ernesto Aparicio-Puerta2Michael Hackenberg3D. Michiel Pegtel4Amsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Pathology, 1081 HV Amsterdam, the Netherlands; Cancer Center Amsterdam, Imaging and Biomarkers, 1081 HV Amsterdam, the Netherlands; Corresponding authorAmsterdam 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 NetherlandsDepartment 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, SpainDepartment 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, SpainAmsterdam UMC, Location Vrije Universiteit Amsterdam, Department of Pathology, 1081 HV Amsterdam, the Netherlands; Cancer Center Amsterdam, Imaging and Biomarkers, 1081 HV Amsterdam, the Netherlands; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723006123BioinformaticsGeneticsGenomicsBiotechnology and Bioengineering |
| spellingShingle | Monique A.J. van Eijndhoven Chantal Scheepbouwer Ernesto Aparicio-Puerta Michael Hackenberg D. Michiel Pegtel IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution Bioinformatics Genetics Genomics Biotechnology and Bioengineering |
| title | IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution |
| title_full | IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution |
| title_fullStr | IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution |
| title_full_unstemmed | IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution |
| title_short | IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution |
| title_sort | isoseek for unbiased and umi informed sequencing of mirnas from low input samples at single nucleotide resolution |
| topic | Bioinformatics Genetics Genomics Biotechnology and Bioengineering |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723006123 |
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