Development and validation of an integrated single-tube whole-genome amplification and library construction system for genomic selection in bovine preimplantation embryos

Genomic selection technology has significantly shortened the generation interval of large single-offspring animals and accelerated genetic improvement, but its application at the early embryonic stage still faces the high risk of embryonic micromanipulation damage, the high cost of whole genome amplification, and the complex and time-consuming workflow. To address these limitations, this study established an integrated whole genome amplification system (named iSALC). The system combines universal primers with specific adapter sequences, allowing the primers to bind to the complementary regions of the adapters, thereby enabling simultaneous amplification and library construction in a single PCR reaction. In terms of sample handling, the system uses phosphate-buffered saline to preserve bovine blastocyst trophoblast cells, with a minimum starting quantity as low as 5 cells. It is recommended to maintain at least 3× sequencing depth to achieve stable coverage uniformity. Whether it is fresh embryo transfer or frozen embryo transfer, pregnancy rates show that embryo micromanipulation not only does not reduce the embryo's implantation ability, but can actually increase the pregnancy rate after embryo transfer. Compared with traditional kits, this system optimized the workflow, eliminated the separate library preparation step, and offered high-throughput compatibility and cost-effectiveness. Overall, this study successfully overcame the dual limitations of early embryo whole genome amplification in terms of cost and operational complexity.