Primary Template-directed Amplification or PTA employs controlled reaction parameters to reproducibly recover greater than 95% of the genome of single-cells and severely limited input samples with best-in-class fidelity and uniformity. 

Discover the difference BioSkryb Genomics can make for you in WGA

tile_balls.jpg

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.

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.

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

PNAS-small.png

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

We provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. 

High-Quality-Nuclei-Isolation-Pub-Thumbnail.webp

High-Quality Nuclei Isolation from Postmortem Human Heart Muscle Tissues for Single-Cell Studies

Featured Resources

## Why work with an incomplete toolbox when you can routinely apply single-cell genomics in cardiovascular disease research?

<div class="float-end right-image-cms">![cardiology-1.png](https://bioskryb-dev-assets.s3.amazonaws.com/uploads/cardiology_1_11ca4ee614.png)</div>

Cardiovascular diseases (CVDs)—including coronary (ischemic), congenital, hypertensive, and rheumatic heart disease, cardiomyopathy, arrhythmia, and other diseases of the heart and blood vessels—are common, and are the leading cause of death and disability worldwide.

Although associated with known behavioral, environmental, and socioeconomic risk factors, a substantial subset of CVDs is directly caused by gene mutations, or are modified by genetic variation. Multi-omics and single-cell analyses are becoming increasingly important in the elucidation of the genetic basis of CVD. Whilst single-cell transcriptomic and epigenomic methods are well established, the application of single-cell genomics in cardiology has been hampered by the limitations of whole-genome amplification ([WGA](/products/)) technologies.

BioSkryb’s robust and scalable WGA technology ([PTA](/technology/)) and powerful bioinformatics platform ([BaseJumper™](/basejumper/)) enables the integration of single-cell genomics in routine clinical and translational cardiology research. With PTA, it is possible to sequence individual cardiomyocytes to detect autonomous cell genetic defects/variation, and genetically profile endothelial cells and fibroblasts in the microenvironment potentially impacts cardiomyocytes. As such, it enables us to utilize the full toolbox of next-generation omics methodologies to support the evolution of personalized management practices for CVDs.

Cardiology

Why work with an incomplete toolbox when you can routinely apply single-cell genomics in cardiovascular disease research?

Featured Resources