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Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures

Nature Protocols volume 13, pages 5978 (2018) | Download Citation

Abstract

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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Acknowledgements

The authors thank J. de Ligt for his input on the CNV analysis, and J.F. van Velzen for his input on the FACS procedures. This study was financially supported by a Zenith grant from the Netherlands Genomics Initiative (935.12.003) and funding from the NWO Zwaartekracht program Cancer Genomics.nl to E.C., and funding from Worldwide Cancer Research (WCR, grant no. 16-0193) to R.v.B.

Author information

Author notes

    • Ruben van Boxtel

    Current address: Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

    • Myrthe Jager
    •  & Francis Blokzijl

    These authors contributed equally to this work.

Affiliations

  1. Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

    • Myrthe Jager
    • , Francis Blokzijl
    • , Sander Boymans
    • , Roel Janssen
    • , Nicolle Besselink
    • , Ruben van Boxtel
    •  & Edwin Cuppen
  2. Hubrecht Institute for Developmental Biology and Stem Cell Research, KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

    • Valentina Sasselli
    •  & Hans Clevers

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Contributions

M.J., F.B., H.C., R.v.B. and E.C. wrote the manuscript. M.J., R.v.B. and V.S. developed the wet lab protocol, and N.B. tested the protocol. The bioinformatics pipeline was developed by F.B. and R.v.B., implemented by F.B. and tested by S.B. and R.J.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ruben van Boxtel or Edwin Cuppen.

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https://doi.org/10.1038/nprot.2017.111

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