Sureplex wga kit

According to the manufacturer's protocol, the biopsied TE cells were lysed and the whole genome was amplified using a SurePlex WGA Kit (Illumina). For chromosomal analysis with aCGH, the WGA products and control DNA were labeled with Cy3 and Cy5 fluorophores according to the manufacturer's instructions and hybridized on 24sure + arrays (Illumina). For chromosomal analysis with NGS, the WGA products were used for library construction with the VeriSeq DNA Library. According to the manufacturer's protocol, NGS was performed on a MiSeqDx instrument (Illumina) using MiSeqDx Universal Kit 3v (Illumina). All results were analyzed using BlueFuse Multi analysis software (Illumina) for chromatin loss or gain across all 24 chromosomes.

The karyotypes in the ART pregnancies were determined by sWGS by NGS. Genomic DNA was extracted from chorionic villi or fetus tissue using Gentra Puregene Tissue Kit (Qiagen Inc., Valencia, CA), in accordance with the manufacturer's protocol. Genomic DNA samples were then diluted to 1ng/μl for whole‐genome amplification (WGA). Subsequently, WGA was performed using the SurePlex WGA Kit (Illumina, San Diego, CA) in accordance with the manufacturer's instructions. Nextera libraries were prepared from the amplified DNA and subsequently sequenced with a VeriSeq PGS assay system by MiSeq (Illumina). The sequencing data were analyzed using BlueFuse Multi analysis software v4.5.

Karyotyping of all samples was performed at OVUS Co., Ltd. (Aichi, Japan). Whole genome amplification was performed using the SurePlex WGA Kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Nextera libraries were prepared from the amplified DNA and subsequently sequenced with a VeriSeq PGS assay system by MiSeq (Illumina, San Diego, CA). The sequencing data were analyzed by BlueFuse Multi analysis software v4.5. The karyotype of each embryo at pre- and post-culture was determined as “euploid,” “mosaic,” or “full aneuploid.” Because single TE biopsy is not sufficient to decide correct karyotype of embryo (Takahashi et al., 2021 (link)), our decision was based on their uniformity when multiple samples were simultaneously collected from the same embryo. When all results were “euploid” or the same “aneuploidy,” the embryo was determined as “euploid” or “full aneuploid.” When they did not match, the embryo was determined as “mosaic.” When the mosaicism rate was between 20% and 80% for a single sample, karyotype of the sample was determined to be mosaic. We calculated the ratios of each karyotype at pre- and post-culture separately to evaluate the change of karyotype during in vitro culture.