Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures
Open accessAbstract
Characterization of mutational processes in adult stem cells (ASCs) will improve our understanding of aging-related diseases, such as cancer and organ failure, and may ultimately help prevent the development of these diseases. Here, we present a method for cataloging mutations in individual human ASCs without the necessity of using error-prone whole-genome amplification. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for accurate whole-genome sequencing (WGS) analysis. We developed a data-analysis pipeline that identifies with high confidence somatic variants that accumulated in vivo in the original ASC. These genome-wide mutation catalogs are valuable resources for the characterization of the underlying mutational mechanisms. In addition, this protocol can be used to determine the effects of culture conditions or mutagen exposure on mutation accumulation in ASCs in vitro. Here, we describe a protocol for human liver ASCs that can be completed over a period of 3-4 months with hands-on time of ∼5 d.
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We included all variants in each clone at autosomal or X chromosomes, not present in the bulk control sample that passed VariantFiltration, with a GATK phred-scaled quality score ≥60; minimum base coverage of 5X, a mapping quality ≥30, and a variant allele frequency of at least 0.15.
“We included all variants in each clone at autosomal or X chromosomes, not present in the bulk control sample that passed VariantFiltration, with a GATK phred-scaled quality score ≥60; minimum base coverage of 5X, a mapping quality ≥30, and a variant allele frequency of at least 0.1558,74.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [74]
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References 53 · Cited by 66
- Authors
- Jager, M., Blokzijl, F., Sasselli, V., Boymans, S., Janssen, R., Besselink, N., Clevers, H., van Boxtel, R., & Cuppen, E.
- Affiliations
- Utrecht University (NL)
- Royal Netherlands Academy of Arts and Sciences (NL)
- Corresponding author
- Cuppen, Edwin
- ORCID
- Venue
- Nature Protocols
- Published
- 2017-12-07
- Volume
- 13
- Issue
- 1
- Pages
- 59-78
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2774520815
- Cited by
- 66
- References
- 53
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 11 · 2023: 8 · 2022: 6 · 2021: 13 · 2020: 5 · 2019: 11 · 2018: 4
- FWCI
- 1.84
- Type
- journal article
- Language
- en
- Open access
- green
- dspace.library.uu.nl
- Licence
- other-oa
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- KNAW Research Portal (The Royal Netherlands Academy of Arts and Sciences) (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Utrecht University Repository (Utrecht University) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- adult stem cells, somatic mutations, mutation accumulation, organoid culture, single-cell genomics, whole genome sequencing, mutational mechanisms, liver stem cells, aging, cancer, mutagen exposure, culture conditions
- MeSH terms
- Mutation Accumulation, Whole Genome Sequencing, Cells, Cultured, DNA, Humans, Liver, Mutation, Organoids, Adult Stem Cells, High-Throughput Nucleotide Sequencing
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