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Retrospective cell lineage reconstruction in humans by using short tandem repeats.

ABSTRACT: Cell lineage analysis aims to uncover the developmental history of an organism back to its cell of origin. Recently, novel in vivo methods utilizing genome editing enabled important insights into the cell lineages of animals. In contrast, human cell lineage remains restricted to retrospective approaches, which still lack resolution and cost-efficient solutions. Here, we demonstrate a scalable platform based on short tandem repeats targeted by duplex molecular inversion probes. With this human cell lineage tracing method, we accurately reproduced a known lineage of DU145 cells and reconstructed lineages of healthy and metastatic single cells from a melanoma patient who matched the anatomical reference while adding further refinements. This platform allowed us to faithfully recapitulate lineages of developmental tissue formation in healthy cells. In summary, our lineage discovery platform can profile informative somatic mutations efficiently and provides solid lineage reconstructions even in challenging low-mutation-rate healthy single cells.

SUBMITTER: Tao L

PROVIDER: S-EPMC8313865 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others.

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Retrospective cell lineage reconstruction in humans by using short tandem repeats.

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