Validation study of orchid's whole genome screening preimplantation genetic testing

Whole genome screening is currently not available in the preimplantation genetic testing (PGT) setting due to poor performance of whole genome amplification (WGA) methods. We aim to validate and offer a comprehensive screening option, which includes PGT for aneuploidy (PGT-A) with improved resolution to cover microdeletion syndromes, PGT for monogenic conditions (PGT-M) without parental probes, and the option of screening for hundreds of severe monogenic diseases simultaneously. DNA from Coriell cell lines and human embryo biopsies were amplified using a custom protocol, sequenced at a depth of 30x using Illumina’s next generation sequencing (NGS) platform and analyzed using GATK4 best practices. Whole genome validation was performed by analyzing five amplified Genome in a Bottle (GIAB) samples, and comparing amplified embryo biopsies results to those on the whole embryo. Our WGA protocol demonstrated consistent and uniform amplification across the entire genome with high amplification success rates (>99.9% for cell lines and >96.2% for embryos). To validate our PGT-A performance, we tested 126 cell lines, as well as 104 replicates from 35 embryos. We validated detection for triploidy, consanguinity, uniparental heterodisomy (down to 5 Mb), and isodisomy calling including complete molar pregnancies. We achieved 100% replication of reporting known pathogenic microdeletions/duplications down to 400 kb (e.g., DiGeorge syndrome and William-Beuren Syndrome). The accuracy, specificity, sensitivity, and precision of the amplified GIAB samples (99.9%, 99.99%, 98.0%, and 98.1%, respectively) were comparable to results on genomic GIAB samples. The accuracy of single nucleotide variant (SNV) calls between biopsies and their source whole embryo achieved high accuracy (99.99%) specificity (99.99%), sensitivity (98.1%) and precision (98.0%). Likewise, for mitochondrial heteroplasmy (99.9%, 100%, 99.1%, and 100%, respectively). To further assess our assay's variant performance, we analyzed 51 samples obtained from both Coriell cell lines and embryos that had been previously submitted for PGT-M analysis. Our assay demonstrated a 100% detection rate for single nucleotide polymorphisms located in crucial genes, including BRCA1, CFTR, and ABCD1. We have validated a WGA protocol for PGT with high accuracy for aneuploidy calls (>99.9%) and SNV calls (99.99%). We validated our detection of microdeletion and microduplications >400 kb as well as our performance of PGT-M without the need of parental probes. This technology has the potential to enable the simultaneous identification of hundreds of pathogenic variants in genes known to be linked to monogenic diseases.