| Whole genome amplification(WGA) is a technology designed to amplify small amounts of DNA down to the single cell level and generate a representative DNA template sufficient for downstream genetic analysis using conventional molecular techniques. A variety of WGA methodologies have evolved but are essentially based on principles of primer extension PCR(PEP), degenerate oligonucleotide primed PCR(DOP-PCR) or multiple displacement amplification(MDA). Applications of WGA are widespread in clinical practice, including genetic typing of circulating tumour cells, identification of forensic samples, and chromosome and single gene analysis of embryos from patients undergoing assisted reproduction and pre-implantation genetic screening(PGS). In the field of PGD/PGS, array-based comparative genomic hybridization(CGH) and single-nucleotide polymorphism(SNP) arrays are widely used to discriminate between euploid and aneuploid embryos in the cohort produced by assisted reproductive technologies. More recently, several next-generation sequencing(NGS) protocols have been developed and validated. Both array and NGS methodologies rely solely on a pre WGA step to amplify the small amount of DNA in an embryo biopsy sample with high fidelity, in order to generate sufficient embryo DNA for either hybridization or sequencing.Currently, the WGA kits that are used in PGD/PGS are commercially available, and have been specifically optimized for single cell application. However, no WGA method has been reported with the capacity to replicate the entire single cell genome. Further, from the analysis of the sequences that are amplified by WGA, there is a substantial bias due to the cumulative effects ofallelic dropoutor preferential allelic amplification. To further improve NGS-based technologies for comprehensive PGD/PGS of the full spectrum of chromosomal abnormalities afflicting human embryos, the WGA reaction remains the most critical step that determines the overall diagnostic performance of both array and sequencing based methods.In this study, we examined the performance of two PCR based methods Sure Plex and MALBAC as well as the MDA based method REPLI-g for genome coverage and bias, and their potential as a starting template for detection of copy number variation(CNV) using our recently described NGS method called CNV-Seq. Here, we showed that both PCR based WGA methods used in conjunction with CNV-Seq, are highly effective for identification, delineation and quantitation of small CNVs in single cells.Single cell fluorescent in situ hybridization(FISH) was initially developed for pre-implantation genetic screening(PGS). The limitations of FISH to simultaneously analyse all 24 chromosomes was first solved by the development of whole genome amplification(WGA) methods such as degenerate oligonucleotide primer PCR in conjunction with single cell metaphase comparative genomic hybridization(CGH).More recently, the development and clinical implementation of alternative diagnostic technologies such as array CGH, single nucleotide polymorphism(SNP) arrays and multiplex quantitative fluorescent-PCR has facilitated widespread testing of embryos for aneuploidy and structural abnormalities that can involve any of the 24 chromosomes.Detailed molecular studies of pre-implantation embryos have shown that whole chromosome aneuploidies originating from meiotic I and II errors and post fertilisation mitotic errors are common, leading to either embryonic growth arrest, implantation failure or spontaneous miscarriage in the early first trimester. In clinical practice, array CGH and SNP arrays generally exhibit high reliability and accuracy for detection of many types of chromosomal abnormalities. However, based on the currently available commercial array platforms, there are significant differences in probe design, probe customization and chromosome probe density distributions, and combined with the potential occurrence of random allelic dropout, the resolution may not be sufficient on some platforms to detect all types of clinically significant chromosomal aberrations in IVF embryos. In addition, in regard to SNP arrays, there is a limitation in calling trisomies and other duplications that originate without meiotic recombination. Further, from biopsy to report, array workflows are still tedious requiring DNA labelling and a long hybridization step. Moreover, array slides and reagents are moderately expensive and the test is difficult to scale efficiently due to limitations in the number of arrays printed per slide. The advent of next generation sequencing(NGS) combined with powerful bioinformatics algorithms to analyse large data sets of mapped sequencing data, opens up new opportunities in PGD/PGS to develop a more comprehensive and cost-effective single platform technology for the diagnosis of the full spectrum of chromosomal abnormalities that occur in IVF embryos.For the study reported here, CNV-Seq was specifically modified for the analysis of embryo biopsy samples by first introducing a whole genome amplification(WGA) step into the sequencing pipeline to generate sufficient DNA template for molecular analysis and then applying smoothing algorithms to the sequencing data to normalise genome bias that is known to be associated with single cell WGA. In embryo validation studies, we assessed the performance of CNV-Seq on WGA products from single embryonic blastomeres with known and unknown chromosome aneuploidies previously analysed by array CGH and demonstrated a high diagnostic concordance between the two techniques. Based on the successful embryo validation phase, we translated the CNV-Seq technology to the clinical diagnosis of embryos from three patients at high genetic risk, and report the first normal PGS pregnancy that has been confirmed by both non-invasive and invasive prenatal diagnosis.Objective 1. We examined the performance of two PCR based methods Sure Plex and MALBAC as well as the MDA based method REPLI-g for genome coverage and bias, and their potential as a starting template for detection of copy number variation(CNV) using our recently described NGS method called CNV-Seq. 2. We speculated that a combination of next generation sequencing technologies and sophisticated bioinformatics would deliver a more comprehensive and accurate methodology to improve the overall efficacy of embryo testing.Methods 1. Using low template DNA(15 pg) and single cells, we compared the two PCR-based WGA systems Sure Plex and MALBAC and REPLI-g WGA MDA system 2. We developed a high-resolution copy number variation(CNV) sequencing pipeline suitable for single cell analysis.Results 1. Using low template DNA(15 pg) and single cells, we showed the two PCR-based WGA systems Sure Plex and MALBAC are superior to the REPLI-g WGA MDA system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. 2. In validation studies, we showed that CNV-Seq was highly sensitive and specific for detection of euploidy, aneuploidy and segmental imbalances in 25 WGA samples from PGS embryos that were originally diagnosed by gold standard array CGH. 3. In clinical translation studies, CNV-Seq was applied as the primary diagnostic method in three clinical PGS cases involving patients at high genetic risk, enabling the identification of euploid embryos for transfer. Embryo transfers resulted in an normal pregnancy that was confirmed by non-invasive prenatal diagnosis and deliveried ahealthy baby girl. CNV-Seq offers a reliable, accurate and cost-effective embryo test to further expand the clinical utility of PGS.Conclusion 1. The main finding from the first study was that PCR based WGA of low template DNA, including 15 pg and one cell samples, provided a far superior template than MDA based WGA for CNV-Seq to identify and correctly delineate pathogenic CNVs. 2. CNV-Seq offers a reliable, accurate and cost-effective embryo test to further expand the clinical utility of PGS. |
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