Bluefuse multi software

Tubing and NGS analysis of cell specimens obtained through TE biopsy were performed according to the method described by Takeuchi et al.²¹ Briefly, as a preliminary preparation, we entered the sample number in the dish (Falcon 351007) to wash the sample cells and a 0.2‐ml tube (Eppendorf polymerase chain reaction [PCR] tubes 0.2 ml) to store the sample. The Pasteur pipette tip was lightly washed with polyvinyl pyrrolidone (Origio) to prevent cell stickiness. After washing the sample three times with phosphate‐buffered solution, it was moved to the bottom of the 0.2‐ml tube, and the tube lid was closed. After centrifugation, the samples were stored at −20°C. The actual NGS procedures were as follows: First, the whole sample was subjected to whole‐genome amplification using a Veriseq PGS kit (Illumina) and a thermal cycler (Mastercycler Nexus, Eppendorf); the obtained sample was quantified and diluted to 0.2 ng/μl, and the diluted sample was amplified using a docosahexaenoic acid tag and PCR. After amplification, cleanup and normalization were performed, as well as load library formation, pooling and loading. The next day, the obtained data were subjected to chart analysis using BlueFuse Multi Software (Illumina).

The STK11 gene is located at 19p13.3. The range of 2 M upstream and downstream of the gene is selected as the main analysis area to determine the informative SNP. Informative SNPs are the main basis for judging which chromosome the embryo inherits from its parent. SNPs that can be used for linkage analysis need to meet the following conditions: (i). The proband's allele is a homozygous genotype at this locus. (ii). The allele of the PJS patient in parents was heterozygous, and the other was homozygous. Combining the SNP data of the proband’s genome NGS and the results of the linkage analysis of the karyomap chip, we constructed haplotypes related to the STK11 mutation and inferred whether the embryo carries a chromosome containing the STK11 mutation. Simultaneously, the original SNP data of the chip and BlueFuse Multi software (Illumina) were used to identify the whole-genome copy number variation (CNVs) of blastocysts. The results of the B-allele frequency and log R ratio charts were based on strict criteria to determine whether there was aneuploidy. At least two laboratory technicians reviewed all the steps.

Single nucleotide polymorphism-array (SNP-array) analysis was performed on DNA of proband 1 and his parents using Infinium CytoSNP-850 K BeadChip (Illumina, San Diego, CA, USA) and in accordance with the manufacturer’s instructions. Array scanning data were generated by iScan system (Illumina, San Diego, CA, USA) and the results were analysed by Bluefuse Multi software (v 4.4).

The amplified WGA products were assessed by aCGH testing with 24sure V3 microarray (Illumina, Inc.) at the Genesis Genetic Asia Laboratory (Taiwan, Taipei). The products and reference DNAs were labeled with Cy3 and Cy5 fluorophores using random primers for 2–4 h. Then the labeling mixes were combined and co-precipitated with COT Human DNA in preparation for hybridization. Labeled DNAs were re-suspended in a dexsulphate hybridization buffer and hybridized onto the 24sure chip for 12 h. Thereafter, the chips were washed and dried. A laser scanner was used to read the resulting images, and BlueFuse Multi Software (Illumina, Inc.) was used to analyze the scan data. More details of aCGH testing procedure can be found in Huang et al., 2013 [34 (link)]. Once a specific amplification was observed, autosomal profiles were analyzed for gain or loss whole chromosomal ratios, using a 3 x SD assessment, greater than ± 0.3log2 ratio call, or both. For hybridization quality control, female samples hybridized with a male reference (sex mismatch) had to show a consistent gain of chromosome X and a consistent loss of chromosome Y [35 (link)].

The PGT-A protocol was similar to our previous report [33 (link)]. In brief, genomic DNA was extracted and amplified using the SurePlex DNA Amplification System (Illumina, San Diego, CA, USA). The amplified DNA product was used to prepare the genomic DNA libraries according to the VeriSeq PGS workflow (Illumina, USA). BlueFuse Multi Software (Illumina, USA) was used for data analysis, and the diploid–aneuploid levels of each sample were examined by at least two technicians. Based upon diploid–aneuploid mosaic ratios measured using the hr-NGS platform for biopsied cells [36 (link),37 (link),38 (link)], blastocysts were classified into the following three groups: (i) euploid blastocysts with mosaicism levels ≤ 20%; (ii) mosaic blastocysts with mosaicism levels between 20% and 50%; and (iii) aneuploid blastocysts with mosaicism levels > 50%.

For small nucleotide variations, pathogenic and likely pathogenic variants as well as VUSs needing parental study were examined using Sanger sequencing on a 3730 DNA Analyzer with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequencing data were aligned against appropriate reference sequences and analyzed using the Sequencher 5.3 software (Gene Codes Corp., Ann Arbor, MI, USA).
Large exonic deletions and duplications were confirmed using the MLPA kit (MRC Holland). Chromosomal copy number alterations were confirmed using the Infinium CytoSNP 850 K array (Illumina) and BlueFuse Multi software (Illumina).

The Sureplex amplified dsDNA products (0.2 ng/μl) were prepared for the Veriseq library via the following steps: first, the dsDNA products were fragmented and then tagged with dual index primers using limited thermal cycles; next, the fragmented and tagged products were purified with AMPure beads via size selection (AMPure XP beads, Beckman Coulter, Pasadena, CA, United States) and normalized to equal concentration in individual sample; then, the final products were pooled to be sequenced using a Veriseq PGS kit (v.3) on a Miseq System (Illumina). Finally, we conducted copy number analysis using BlueFuse Multi Software (Illumina). The samples were distinguished if a median chromosomal copy number deviated from the default line; additional details of the aneuploidy calling regarding determinations of copy number variation were based on work by Fiorentino et al. (2014) (link) and manufacturer’s instructions (Illumina, a technical guide to aneuploidy calling with Veriseq PGS).