AB-PGT™ kits Cat# W02 (for MiSeq) and Cat# W03 (for NextSeq) are sufficient for processing 288 samples.
Kit Cat# W02 is recommended for multiplexing about 36-48 samples for a single MiSeq run, and Cat# W03 for multiplexing up to 144 samples for a NextSeq run.
Most of the components of the kits are the same, except the NextSeq kit has additional indexing primers for more powerful multiplexing.
MiSeq kit Cat# W01 has the same components as Cat# W02, except it contains three times less reagents and is sufficient for processing only 96 samples.
Operating the kits is simple (Fig. 1). DNA is extracted from trophectoderm biopsies (3-10 cells) in a lysis cocktail (A). After the lysis, amplification cocktail is added and whole genome amplification (WGA) reaction (B) is conducted in the same tube. Next, an aliquot of amplified DNA is processed for detecting aneuploidies (PGT-A), and the rest of the sample is stored for detecting monogenic disorders (PGT-M) or as a reference if the need arises.
Fig. 1. Cell lysis and AB-PGT™ WGA workflow.
With minimal manipulations and only ~30 min hands-on time, AB-PGT™ WGA (patent pending) is less prone to contamination than most other methods. Another advantage is exhaustive random priming of the template DNA at the very first WGA cycle, which is crucial for minimizing amplification bias and for improved genome coverage. Therefore, unlike most other WGA methods, that either sacrifice genome coverage for specificity of CNV detection, or accuracy of CNV detection for better genome coverage, this method maintains faithful CNV representation (Fig. 2) with deep genome coverage, thus outperforming competition in locus-specific detection (Fig. 3).
Fig. 2. CNV detection. DNA from thirty-eight biopsies were extracted, amplified and indexed using AB-PGT™ Cat# W02 kit, then pooled and sequenced in a single MiSeq run. The raw data were processed for copy number variations (CNV) using proprietary CNVector™ software. Advanced statistical analysis (not shown) with assigned multi-color scheme facilitates PGT-A report even with biopsies of below average quality.
Typically, the embryos that pass the PGT-A test are suitable for implantation. However, embryos from parents that have a family history of monogenic disorders also have to be tested in PGT-M. It is beyond our scope to provide reagents for PGT-M testing; this would involve validation of hundreds of PCR primers targeting disease-associated loci. However, a model experiment indicates that DNA amplified in AB-PGT™-WGA is quite suitable for PGT-M analysis (Fig. 3).
Fig. 3. Locus specific detection, PGT-M model experiment. Five human cells were sorted into each well of PCR plates and processed using ether AB-PGT™ W02 kit or a competitor kit (current leader in PGT testing). The WGA products were amplified with 70 pairs of locus-specific primers in QPCR. 1-10 AB-PGT™ kit, 11-20 competitor kit, 21-22 bulk human DNA.
Illumina paired end sequencing. The kits contain a complete set of reagents for processing biopsy samples for sequencing, except AMPure XP magnetic beads (Beckman Coulter) that are necessary for purification of amplified DNA. In addition, MiSeq Reagent Kit V3 (150 cycles) or NextSeq 500/550 Kit v2.5 (75 cycles) have to be purchased from Illumina and used according to the manufacturer’s instructions. Please refer to Illumina documents 15041638 v01 and 15057456 v02 regarding the use of custom primers, 1000000041074 v07 for multiplexing, and to on-line recommendations: “Best practices for manually normalizing library concentrations, Illumina, 08/21/20”.