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.

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.

Current preimplantation genetic testing (PGT) enables the selection of embryos based on fetal aneuploidy or the presence a small number of preselected disease-associated variants. Here we present a new approach that takes advantage of the improved genome coverage and uniformity of primary template-directed amplification (PTA) to call most early embryo genetic variants accurately and reproducibly from a preimplantation biopsy. With this approach, we identified clonal and mosaic chromosomal aneuploidy, de novo mitochondrial variants, and variants predicted to cause mendelian and non-mendelian diseases. In addition, we utilized the genome-wide information to compute polygenic risk scores for common diseases. Although numerous computational, interpretive, and ethical challenges, this approach establishes the technical feasibility of screening for and preventing numerous debilitating inherited diseases.

Treatment of acute lymphoblastic leukemia (ALL) necessitates continuous risk assessment of leukemic disease burden and infections that arise in the setting of immunosuppression. This study was performed to assess the feasibility of a hybrid capture next-generation sequencing panel to longitudinally measure molecular leukemic disease clearance and microbial species abundance in 20 pediatric patients with ALL throughout induction chemotherapy. This proof of concept helps establish a technical and conceptual framework that we anticipate will be expanded and applied to additional patients with leukemia, as well as extended to additional cancer types. Molecular monitoring can help accelerate the attainment of insights into the temporal biology of host-microbe-leukemia interactions, including how those changes correlate with and alter anticancer therapy efficacy. We also anticipate that fewer invasive bone marrow examinations will be required, as these methods improve with standardization and are validated for clinical use.

Whole-genome sequencing of preimplantation human embryos (PGT-WGS) is not currently performed due to the insufficient quality of amplified DNA from embryo biopsies. Here we present a PGT-WGS approach that takes advantage of the improved genome coverage and uniformity of primary template-directed amplification (PTA) to call almost all early embryo genetic variants accurately and reproducibly from a preimplantation biopsy. In a research sibling cohort, we identified clonal and mosaic chromosomal aneuploidy, de novo mitochondrial variants, and variants predicted to cause mendelian and non-mendelian diseases. In addition, we utilized the genome-wide data to compute polygenic risk scores for common diseases. Finally, we performed a clinical study on the parents and 8 sibling embryos of an infant with neonatal-onset seizures and severe developmental delay. We first used low-pass whole genome sequencing to identify embryos most likely to successfully implant based on aneuploidy. We then performed PGT-WGS and evenly covered a mean of 99.1% of the embryo genomes at least 10X, resulting in the detection of 99.5% of parental variants and enabling us to screen genome-wide for inherited and de novo genetic variants in each embryo using ACMG rules. Although numerous computational, interpretive, and ethical challenges remain for widespread implementation, this study establishes the technical feasibility of screening for and preventing numerous debilitating genetic diseases with PGT-WGS.