Abstract
Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.
Highlights
Whole genome amplification (WGA) is required for the unbiased sequencing of minute DNA samples
We found that primary template-directed amplification (PTA) again had the lowest coefficient of variation (CV) and median absolute pairwise distance (MAPD) compared to all other whole genome amplification (WGA) methods at every bin, with the exception of the MAPD of Ampli1 with large bin sizes (Fig. 3G)
To provide more comprehensive high-resolution details of the mutagenicity of compounds in human cells, we developed a system we have named direct measurement of environmental mutagenicity (DMEM) where we expose human cells to an environmental compound, perform highquality single-cell variant calling with PTA, and create a map of mutagen–genome interactions that occurred while each cell was alive
Summary
Whole genome amplification (WGA) is required for the unbiased sequencing of minute DNA samples. We present a method we have named primary template-directed amplification (PTA), which takes advantage of the processivity, strong strand displacement activity, and low error rate of phi polymerase used in MDA. In this method, exonuclease-resistant terminators are incorporated into the reaction, creating smaller double-stranded amplification products that undergo limited subsequent amplification. Applications of measuring genetic diversity from single cells with PTA presented in the manuscript include examining the acquisition of genetic changes during normal development and aging, measuring the consequences of specific perturbations such as genome editing, and characterizing the evolution of clonal populations during cancer formation
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