## Why base a critical decision on selective information when you can rely on complete, genome-wide screening?

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

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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

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_In vitro_ fertilization (IVF) continues to offer hope for millions of families that struggle with infertility. Pre-implantation genetic screening (PGT)—designed to screen embryos for aneuploidies and known genetic variants—is a critical part of the process. 

Molecular techniques based on PCR, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), SNP arrays, or next-generation sequencing (NGS) enable high resolution, accurate genotyping, representing a significant advance over the insights supported by strict phenotypic evaluation of embryos. Of these methods, NGS is the only one capable of offering information about all major types of chromosomal abnormalities, as well as a genome-wide assessment of known variants associated with mendelian and non-mendelian diseases.

BioSkryb’s highly accurate and scalable single-cell whole genome amplification ([WGA](/products/)) technology ([PTA](/technology/)) captures near-complete genomes from minute quantities of genetic material, thereby increasing the sensitivity and precision of PGT. This enables more comprehensive screening of embryos to support life-changing pre-implantation decisions.

Peter L. Nagy MD PhD

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Working with Bioskryb Genomics was an amazing experience. In part because of the quality of the training and support they provided during the assay setup, in part because their method generates gorgeous data. While acting as Director of the Personalized Genomic Medicine Division at Columbia University in the mid 2010s, I experimented with single-cell genome amplification. I quickly gave up on that effort because of the poor uniformity and high artifact content of the PCR-based technologies. Using the Bioskryb Genomics method was really a revelation. The uniformity of amplification and the specificity and sensitivity of both SNV and CNV detection was comparable to unamplified genome sequencing results. This technology represents a great promise for reproductive medicine as well as for other applications. Great contribution to science and medicine; Congratulations to the developers!”

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Chief Medical Officer, Praxis Genomics

BioSkryb Genomics has developed a workflow to enable single-cell whole genome amplification and sequencing analysis. The newly-developed BioSkryb preimplantation genetic testing (PGT) workflow solves a significant workflow challenge by allowing the analysis of gross chromosomal aneuploidy errors (PGT-A) simultaneously with comprehensive monogenic genetic disorder single nucleotide variation analysis (PGT-M). This fully-integrated workflow (Figure 1) highlights the steps; from embryo biopsy at the IVF center through whole genome amplification (WGA), library preparation, sequencing and analysis.  The industry-leading BioSkryb Genomics chemistry (Figure 2) gives laboratories the capability to combine PGT-A and PGT-M in a single PGT workflow from the biopsy of a single embryo.  This allows for whole genome and/or specific panels for known inherited mutations to be leveraged.  Due to the unprecedented completeness of genome coverage by PTA, there is no need for splitting samples, multiple workflows or any other downstream changes to your normal PGT workflow.  The workflow makes this possible from a 4-6 cell embryo biopsy down to a down to single blastomere.

BioSkryb Genomics has developed a workflow to enable single-cell whole genome amplification and sequencing analysis. 

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A Fully Integrated Workflow Based on ResolveDNA® WGA for Complete Analysis of Human Embryos for Aneuploidy and Monogenic Disease (PGT-A + M)

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Genome-Wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification

Whole genome screening is currently not available in preimplantation genetic testing (PGT) due to poor performance of whole genome amplification methods. Here, we present the first validation study using our latest whole genome amplification (WGA) protocol, which yields amplified DNA with comparable quality to genomic DNA when perfoming whole genome sequencing assays. We started by validating PGT for aneuploidy (PGT-A) using our WGA protocol on cell lines and donated human embryos, where amplification success rates were >99.9% and 96.2% respectively. Accuracy on both was >99.9% on aneuploidy status. We next validated whole genome sequencing using amplified and genomic DNA from the Genome in the Bottle reference sample NA12878 to demonstrate accuracy, specificity, sensitivity, and precision of our WGA methods. Amplified DNA and genomic DNA are comparable in this case, with 99.99% accuracy, 99.99% specificity, 98.0% sensitivity and 98.1% precision with our WGA protocol. We additionally examined variant calls between biopsies and the remaining embryos from which they were derived. Again, 99.99% accuracy, 99.99% specificity, 98.1% sensitivity and 98.0% precision were observed. Mitochondrial heteroplasmy between biopsies and embryos was validated, and 99.9% accuracy, 100% specificity, 99.1% sensitivity and 100% precision were observed. Finally, we demonstrated the ability to improve the accuracy of certain types of mendelian variants in our data to close to 100% by leveraging parental and a born child’s genome to perform linkage analysis.

Here, we present the first validation study using our latest whole genome amplification (WGA) protocol.

Reproductive Genetics

Why base a critical decision on selective information when you can rely on complete, genome-wide screening?

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Peter L. Nagy MD PhD