Precise exome analysis of blastocyst biopsy scale samples using primary template-directed amplification

This study evaluates primary template-directed amplification (PTA) for whole exome sequencing (WES) of small fibroblast cell groups, which mimics the limited cell quantities typical of trophectoderm embryo biopsies. PTA’s consistent amplification reduces allelic dropout (ADO) and improves uniform coverage, overcoming challenges associated with conventional methods such as multiple displacement amplification (MDA). Using fibroblast samples alongside well-characterized genomic references (E701, NA12878), we benchmarked PTA-WES, achieving 97.5% target region coverage at 10x, meeting American College of Medical Genetics and Genomics (ACMG) standards. The completed filtering and variant calling provide a foundation for further optimization and analysis aimed at evaluating the reliability of PTA for routine clinical use. Preliminary results from embryo biopsies sequenced with PTA-WES revealed a median coverage of 102x, significantly improving upon the variability and coverage gaps observed with MDA-WES. These findings support the potential of PTA to increase the clinical applicability of WES for preimplantation genetic testing for monogenic disorders (PGT-M), expanding its ability to detect inherited and de novo mutations in embryos.