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. 2020 May 19;11:2493. doi: 10.1038/s41467-020-16323-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic overview of the experimental setup to determine in vitro-accumulated mutations in individual human PSCs, liver stem cells, and intestinal stem cells. Clonal stem cell lines were cultured for ~2–5 months during which mutations were allowed to accumulate. At the end of the culture period, a second clonal step was performed, and the derivative subclones were expanded until enough DNA could be isolated for WGS analysis. A biopsy or bulk culture was used as a reference sample to determine and exclude all germline variants, which were subtracted from the clone (comparison 1) and from the subclone (comparison 2). Finally, with these results, the clonal variants were subtracted to identify all variants that were unique to the subclone. b Growth curves for PSCs (green), liver stem cells (red), and intestinal stem cells (blue). c Box–whisker plots showing the median, first and third quartiles, and minimum and maximum of the number of indels per genome per population doubling (statistical test used: ANOVA). d Box–whisker plots showing the median, first and third quartiles, and minimum and maximum of the number of SBS per genome per population doubling. Statistical test used: ANOVA. e Top panels: genomic distribution of the observed versus the expected amount of mutations per genome per doubling. Bottom panels: log2 of the ratio between the total number of observed mutations versus the total number of the expected amount of mutations. Asterisks denote statistically significant differences between observed and expected (binomial test). CDS coding sequence. f Relative number of SBS per cell type (left circles) and those affecting protein-coding DNA (right circles). Nonsynonymous SBS leads to amino-acid changes, whereas synonymous mutations do not have an effect on the protein sequence. g Top panels: genomic distribution of the observed versus the expected amount of mutations per genome per doubling at promoter and promoter-flanking regions. Error bars represent the standard deviation. Bottom panels: log2 of the ratio between the total number of observed versus the total number of the expected amount of mutations at promoter and promoter-flanking regions. Asterisks denote statistically significant differences between observed and expected.