Next-Generation Sequencing for PGT-A Validation

Next, NGS analysis was used as a validation test in some embryos analyzed. Amplification products were processed, bar-coded, purified, pooled, denatured, and then sequenced to prepare DNA libraries following the manufacturer’s guidelines (VeriSeq PGT-A Illumina, Inc). The MiSeq Reagent Kit v.3 (Illumina, Inc.) was used on a MiSeq System (Illumina, Inc.). The generated bioinformatics data were analyzed by BlueFuse Multi Software (Illumina, Inc.). Embryos were identified by a median chromosomal copy number deviation from the default copy number. Possible trisomy or monosomy of embryo autosomal chromosomes was seen as copy numbers >2 or <2, respectively. Details of preparation procedures and the determination of automated copy number for each chromosome on BlueFuse Multi Software (Illumina, Inc.) were described in Fiorentino et al.’s (6 (link)) and Lai et al.’s study (31 (link)).