Dissecting single-cell genomes through the clonal organoid technique

Abstract

The revolution in genome sequencing technologies has enabled the comprehensive detection of genomic variations in human cells, including inherited germline polymorphisms, de novo mutations, and postzygotic mutations. When these technologies are combined with techniques for isolating and expanding single-cell DNA, the landscape of somatic mosaicism in an individual body can be systematically revealed at a single-cell resolution. Here, we summarize three strategies (whole-genome amplification, microdissection of clonal patches in the tissue, and in vitro clonal expansion of single cells) that are currently applied for single-cell mutational analyses. Among these approaches, in vitro clonal expansion, particularly via adult stem cell-derived organoid culture technologies, yields the most sensitive and precise catalog of somatic mutations in single cells. Moreover, because it produces living mutant cells, downstream validation experiments and multiomics profiling are possible. Through the synergistic combination of organoid culture and genome sequencing, researchers can track genome changes at a single-cell resolution, which will lead to new discoveries that were previously impossible.

Fig. 1. The origin of genomic variants in human cells and their characteristics.

a The origin of genomic mutations together with the cellular phylogeny of an individual’s development. Four different origins (inherited, de novo, and embryonic and late stages) are represented. b The characteristics of genomic variants according to their origin. The mutation number per cell (x-axis) and the proportion of mutant cells in an individual’s body (y-axis) are illustrated. For postzygotic mutations, we assume that the endogenous mutation rate is 1 mutation per cell per cell doubling. The threshold of mutation detection (6.6% of the cell fraction) is calculated with 30-fold whole-genome sequencing. c The signal intensity of genomic variants in ordinary bulk-tissue whole-genome sequencing according to the origin of the genomic variants. WGS, whole-genome sequencing.

Fig. 2. Three strategies for detecting genome-wide mutations in single cells.

Three different strategies that can be applicable for detecting single-cell mutations are illustrated with brief experimental steps. Expected outcomes from each of the strategies are shown below with summaries of source genomes and sequence reads produced from the source. True, borderline, and false findings are highlighted with green, orange, and red boxes, respectively. LCM laser-capture microdissection.

Fig. 3. Applications of the clonal organoid technique.

a By reconstructing cellular phylogenies using somatic mutations as barcodes, clonal dynamics in early development can be traced. b The genome-wide mutational burden and signatures can be investigated in normal and diseased cells when clonal organoids are derived from healthy and diseased tissues. c The functional impact of a specific gene mutation can be investigated via the genetic engineering of organoids followed by sequencing. d By exposing organoids to carcinogens, their mutagenic impacts can be accurately assessed. e Organoid technologies enable multiomics profiling because a large number of progeny cells, which share similar genomic and phenotypic backgrounds, can be produced.