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 â 1/45 d.
Original language | English |
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Pages (from-to) | 59-78 |
Number of pages | 20 |
Journal | Nature Protocols |
Volume | 13 |
Issue number | 1 |
DOIs | |
Publication status | Published - 1 Jan 2018 |
Keywords
- Adult Stem Cells
- Cells, Cultured
- DNA
- High-Throughput Nucleotide Sequencing
- Humans
- Liver
- Mutation
- Mutation Accumulation
- Organoids
- Whole Genome Sequencing