Bluefuse multi

NIPGS, TE and WB samples were processed using the same protocol as described above, assessed for ploidy status (euploid vs. aneuploid) using BlueFuse Multi software (Illumina, CA) and, in the case of aneuploid embryos, the type and size of detected chromosomal aberrations was indicated (Fig 2). Results were considered as fully concordant only when ploidy and chromosomal aberrations fully matched between sample types. Partial concordance was indicated when only some of chromosomal aberrations between samples were in agreement. Results where samples showed no concordance with regards to chromosomal composition were classified as discordant.

Whole genome amplification (WGA) was performed, according to manufacturer’s instructions, using the SurePlex WGA (VeriSeq PGS Kit, Illumina). The WGA starts with enzymatic lysis of biopsied cells or miPGT samples (5 ul SEM + BF) to release gDNA followed by a pre-amplification and amplification steps using degenerative primers for uniform random whole genome amplification.
When a cell lysis step was not performed, WGA SurePlex protocol starts with direct pre-amplification of 10ul miPGT (SEM + BF) sample. WGA products (SurePlex kit, Illumina) were quantified with the Qubit3.0-Fluorometerand their size distribution was assessed using 2100 BioAnalyzer (DNA high sensitivity chip, Agilent).
All samples were diluted to 0.2 ng/ul and a total of 1 ng from each sample and amplified using random primers. The kit contains 24 unique indexes added by amplification. Indexed DNA libraries were cleaned-up (AMPure XP beads 1:1 ratio) and normalized using magnetic beads. The normalized libraries were pooled, denatured, and sequenced using a MiSeq (single-end, 1 × 36 bp). Alignment and demultiplexing are done as part of the VeriSeq PGS protocol on MiSeq and CNV analysis and visualization were done using BlueFuse Multi (Illumina) software. Reporting was done using Hg39 reference with threshold for mosaicism of >30% and CNV changes >10 Mb.

Chromosomal microarray analysis by Agilent 8 × 60 K microarray chip and the NGS by VeriSeq PGS kit were conducted according to the manufacturer’s protocols. Data analysis was performed with Cytogenomics 2.7.8.0 software (Agilent) for CMA and BlueFuse Multi for NGS (Illumina, Inc.). Both pipelines used human genome build 19 (hg19/GRCh37) as a reference genome.
The NGS-based PGT-A method is characterized by having high detection resolution for segmental aneuploidies [19 (link),20 (link)]. Based on in-house validation, the resolution of segmental aneuploidy was set as 10 Mb for both CMA and NGS PGT-A platforms [21 (link),22 (link)]. The level of mosaicism was calculated by the copy number ratio (Equation (1)) and reported with a range of 20% to 80% mosaic level.

Whole genome copy number variation (CNV) analysis was performed by whole genome low pass (0.1×) NGS using the VeriSeq® PGS Kit (Illumina, CA). Briefly, after WGA, according to manufacturer’s instructions, gDNA was tagmented and amplified. The amplified gDNA was indexed, purified using AMPure XP beads (1:1 ratio), and normalized using magnetic beads. The normalized libraries were pooled, denatured, and sequenced using a MiSeq (single-end, 1 × 36 bp). BlueFuse Multi (Illumina, CA) was used for chromosome CNV analysis and data visualization. The optimal metrics for standard clinical analysis of embryo ploidy are 500,000 reads passing filter and a sample noise score (derivative log ratio - DLR) of <0.2; 250,000 reads and DLR < 0.4 is clinically acceptable.

Karyomapping gene chip detection was performed on the embryonic DNA samples after WGA and the peripheral blood samples of the subjects and couples. The specific steps were carried out according to the instructions of the Karyomap chip, including DNA fragmentation, precipitation, resuspension, hybridization, washing, extension, and colouring. Finally, the amplified DNA was scanned on the HumanKaryomap-12 Bead Chips (Illumina) platform, and the results were analysed by BlueFuse Multi (Illumina) software.

24 Sure (+) BAC microarray (BlueGnome, Illumina, USA) was used for single chromosome 6 screening, described by Tobler et al.25 . Briefly, DNA samples were labelled with random primers, hybridised to a BAC microarray with Cy3, scanned on InnoScan 710 (Innopsys) and reference compared against a euploid male labelled with Cy5. Raw fluorescence was measured by Mapix (Innopsys) and signal strength normalised into the presence of chromosomal DNA by Bluefuse Multi (BlueGnome, Illumina, USA).

Embryos were kept in culture in a time-lapse incubator, single-step culture media (LifeGlobal^®, Paramus, NJ, USA), with 5% oxygen concentration, as described previously (17 (link)). Embryos were transferred at the stage of the blastocyst. When performed, the PGT-A procedure was carried out as previously described (23 (link)). Embryos were biopsied and vitrified on days 5 and 6 of development. Genetic testing on the embryo was performed by array comparative genomic hybridization (a-CGH) using accessible kits and software (SurePlex^® DNA Amplification System, 24Sure^® Microarray Pack, BlueFuseMulti^®, Illumina^®) following the manufacturer’s instructions.