Bluefuse multi v4

G-banded chromosome analysis and karyotyping were done by standard methods. Interphase fluorescence in situ hybridization (FISH) analysis was performed on peripheral blood cells from the patient using probes for chromosomes X (DXZ1) and Y (DYZ3). Chromosomal single nucleotide polymorphism (SNP) microarray analysis of genomic DNA prepared from peripheral blood (the patient, his mother, his brother, and one maternal uncle) or from saliva (his sister, another maternal uncle) was performed using the Illumina Infinium CytoSNP-850K BeadChip v1.1. Microarray data was visualized and analyzed using Illumina BlueFuse Multi v4.4.

A genomic array was performed for all 14 patients using the Infinium CytoSNP‐850K BeadChip, which contains approximately 850,000 selected single nucleotide polymorphisms (SNPs) with an enriched coverage for 3,262 disease‐related genes (Illumina, Inc.). The manufacturer's recommended protocol was followed and the raw data were analyzed using BlueFuse^™Multi v4.4 (Illumina, Inc.). The genomic positions are given as mapped to the GRCh37/hg19 genome build.
The results were analyzed according to the American College of Medical Genetics guidelines (Kearney, Thorland, Brown, Quintero‐Rivera, & South, 2011) using independent tests and were compared with the following databanks of CNVs and classified as benign, pathogenic, or VOUS (variants of uncertain clinical significance): the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation/), the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER, http://decipher.sanger.ac.uk/) and the UCSC Genome Bioinformatics database (http://genome.ucsc.edu). The genomic positions are reported according to their mapping on the GRCh37/hg19 genome build.

Genetic workup started with single nucleotide polymorphism–array (SNP-array) analysis using the Infinium CytoSNP-850K v1.2 BeadChip Kit (Illumina) according to manufacturer’s protocol. The BeadChips were scanned using the NextSeq 550 System (Illumina). Data was analyzed with BlueFuse Multi v4.5 (Illumina).

Biopsied samples were subjected to WGA using an REPLI-g Single-Cell WGA kit (Qiagen, Germany) according to the manufacturer's instructions. Embryonic WGA and parental DNA samples were used for SNP genotyping to detect chromosome status and monogenic disease (Wang et al. 2019 (link); Li et al. 2021 ). Briefly, individual DNA samples for PGT-M were hybridized with HumanKaryomap-12 v2.1 BeadChip (Illumina, USA), which contains almost 300,000 SNPs with an average distance of 9.7 kb (Wang et al. 2019 (link)). BlueFuse (Multiv 4.5; Illumina) was used to process data files. Haplotypes were generated by reformatting and importing genotype data into an in-house transcript. Simultaneously, aneuploidy was detected visually based on the B-allele frequency of SNPs and the logR ratio defined by Illumina, with normal DNA as a reference (Wang et al. 2019 (link)). The detailed steps of haplotype phasing were described previously (Wang et al. 2019 (link)). For PGT-SR, individual DNA samples were hybridized with HumanCytoSNP-12 v2.1 BeadChip (Illumina; Li et al. 2021 ). The breakpoints of reciprocal translocation were identified using unbalanced embryos, as we described previously (Li et al. 2021 ). Based on the deletion position of translocated chromosomes in unbalanced embryos generated by the adjacent mode, the specific location of the breakpoint could be determined (Li et al. 2021 ).

Genomic DNA was isolated from the peripheral blood samples using the Maxwell RSC Blood DNA kit (Promega) as per the manufacturer’s instructions. Comparative genomic hybridization (CGH) assays were conducted with the SurePrint HD array (2 × 400 K V.1.0, Agilent Inc.), which was designed to detect CNVs across the whole genome. Of note is that the array contained 400 K oligonucleotides with a median probe spanning of ∼5.3 kb that represented the coding and the non-coding sequences in the human genome. The DNA samples from healthy individuals were used as controls for data analysis. We analyzed the CGH array data using the CytoGenomics software provided by Agilent.
Whole-genome single nucleotide polymorphism (SNP) array tests were executed by utilizing the Infinium CytoSNP-850K kit (v1.1 BeadChip, Illumina), following the manufacturer’s guidelines. Specifically, the Illumina chip contained nearly 850,000 empirically selected SNPs spanning the whole genome. The average inter-probe distance was approximately 1.8 kb, and the overall effective resolution was around 18 kb. The processed chip was scanned on the NextSeq550 system (Illumina). The data were analyzed using the BlueFuse Multi v4.3 software (Illumina).
Cell preparation and karyotype analysis were performed as previously reported (Kim et al., 2011 (link)). For each patient, 20 metaphase cells were analyzed by G-banding.