Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification

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), a new 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 new types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a new 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 whole genome amplification, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control

“…Applying this approach across cell types, genetic backgrounds, and experimental conditions will reveal how replication is altered at the spatiotemporal levels in different physiological contexts. With constantly improving methods for high-throughput single cell isolation and accurate whole-genome amplification 18,19,30 , this approach promises to become ever more informative for the understanding of the DNA replication timing program.…”

“…Previous sequencing-based studies measured DNA replication timing in a relatively small number of cells, mostly limited to mid-S phase cells 15,16 . To analyze single cells, these studies performed DNA amplification using DOP-PCR, which is known to yield suboptimal DNA copy number measurements 18,19 . Consequently, these studies were limited to analyzing replication timing at the level of large chromosomal domains (typically on the order of megabases).…”

“…As an alternative approach, we devised a method to study DNA replication timing across the entire span of S phase, in hundreds to thousands of cells, and with higher spatial resolution than previous methods. Specifically, we used the 10x Genomics Single Cell CNV platform, a microfluidics platform for isolation, barcoding, and multiple-displacement amplification (MDA; which has been shown to be superior to DOP-PCR 18 ) of single cells, which was then followed by whole-genome sequencing. As an initial proof-of-principle, we analyzed 5,793 cells isolated and sequenced from the human lymphoblastoid cell line (LCL) GM12878 following fluorescenceactivated cell sorting (FACS) of G1-and S-phase cells.…”

Ultraspecific somatic SNV and indel detection in single neurons using primary template-directed amplification

“…sSNV false discovery rate estimation Estimated FDR curves shown in Figure 2a were parameterized by FDR = FP rate per Mb FP rate per Mb + Sensitivity × Mutations per Mb Parameters used were: PTA with GATK (ref. 15), FP rate per Mb = 0.9, sensitivity = 0.8; PTA with SCAN2 (multi-sample calling) FP rate per Mb = 0.0143, sensitivity = 0.457 (derived from simulation experiments, see Synthetic diploid simulations). To compute the best-case scenario for MDA, we assumed that all artifacts caused by single stranded dropout would be erroneously identified as true SNVs and that these would be the only source of FPs.…”

“…We previously used read-level phasing to filter artifacts in MDA samples and discovered an age-associated increase in somatic mutations in human neurons 6 , but were limited to analyzing mutations within a few hundred base pairs of germline SNPs (~15% of the genome). A newly developed single-cell amplification method called primary template-directed amplification (PTA) aims to reduce these artifacts by dampening the exponential nature of isothermal MDA 15 .…”