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Clinical embryo sample tracking and identification using the mitochondrial genome
The mitochondrial genome contains single nucleotide variants (SNVs) that can be used to differentiate individuals, and are routinely used for population genetic studies and ancestry. Mitochondrial DNA (mtDNA) is maternally inherited, providing a novel opportunity for DNA-based confirmation of maternal origin of embryo biopsies and sibling embryo identification. The mitochondrial genome is sequenced during Preimplantation Genetic Testing for Aneuploidy (PGTA) by Next Generation Sequencing (NGS) and the depth and breadth of coverage obtained from PG-Seq™ readily allows SNV analysis, even from a 48 sample NGS run. This readily available information could be used for sample tracking within an IVF or genetic service provider laboratory.
Aim – To demonstrate the use of the RHS Embryo ID panel to achieve accurate and economical embryo identification as part of routine PGT-A using PG-Seq™.
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A novel single tube amplification and barcoding approach for PGT-A using Ion Torrent™ NGS
There are several Whole Genome Amplification (WGA) and library preparation methods to prepare embryo biopsies for Next Generation Sequencing (NGS) for Preimplantation Genetic Testing for Aneuploidy (PGT-A). DOPlify® provides a flexible technology to not only amplify whole genomes but also target sequences using RHS’ Target Sequence Enrichment (TSE) protocol. The attributes of DOPlify® also present a unique mechanism to incorporate the specific sequences needed for NGS. The incorporation of PCR barcoding during WGA provides laboratory efficiencies compared to sequential WGA followed by NGS library preparation, including a reduction of hands on and total protocol time and decreased reagent requirements for sample preparation.
Aim – To develop a novel approach that allows amplification and PCR barcoding of samples in a single tube for Ion Torrent™ NGS.
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DOPlify®, Target Sequence Enrichment and Allele Drop Out – is there a benefit?
Allele Drop Out (ADO), or failed PCR amplification of one or both alleles, presents a significant risk of misdiagnosis or the return of a “no result” for PGT-M cycles. Although a PGT-M result may indicate that an embryo is unaffected by a monogenic disease, standard PGT-M methods are unable to assess aneuploidy and up to 50% of these transferred embryos may be aneuploid1. Combining Whole Genome Amplification (WGA) along with gene-specific primers in a single PCR reaction to allow both aneuploidy detection (PGT-A) and monogenic disease detection (PGT-M) is an advantage of DOPlify® and RHS’ Target Sequence Enrichment (TSE) protocol.
Aim – To compare ADO rates across three different approaches; Gene Specific PCR only (current PGT-M methodology), DOPlify® WGA only (current PGT-A methodology) and DOPlify® WGA with TSE (DOPlify® PGT-A with RHS’ TSE patented approach).
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Optimisation of whole genome amplification specifically for non-invasive PGT-A using spent embryo culture media
The clinical use of non-invasive preimplantation genetic testing for aneuploidy (PGT-A) requires concordance of the spent embryo culture media result to the embryo biopsy result and the ability to distinguish maternal contamination from the embryonic DNA, especially for a euploid female result. Although concordance of spent embryo culture media and trophectoderm biopsy has been reported at as high as 95% following the collection of samples at Day 5-7 using DOPlify® (Lane et al, 2017), the ability to test media collected earlier in culture requires an increased level of sensitivity. Additionally, there are a number of known PCR inhibitors in culture media, including salts and proteins, which need to be overcome.
Aim – To identify an optimal protocol to amplify DNA in spent embryo culture media that maximises WGA DNA yield and NGS results.
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WGA and NGS read length significantly impact mitochondrial characterisation
The mtDNA genome is 16,571bp in length and there are multiple copies per cell. It contains mutations linked to diseases such as cancer, diabetes and deafness. These attributes make mtDNA an ideal model to evaluate performance metrics of whole genome amplification (WGA) technologies.
Aim - This study aimed to compare two different commercially available WGA kits using short and mid-range read length NGS; evaluating overall mtDNA genome coverage along with coverage of 23 common mitochondrial mutations.
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PG- Seq™ - an accurate complete NGS solution for PGS
PG-Seq™ for Pre-implantation Genetic Screening developed by RHS Ltd offers a novel complete, cost effective workflow. The product includes DOPlify™ lysis and whole genome amplification reagents, latest generation NGS library preparation reagents and easy to use data analysis software all compatible with the Illumina MiSeq sequencer. The product has been developed for the analysis of up to 48 embryo biopsies in a single NGS run, twice the capacity of VeriSeq, with no change to equipment required.
Aim – To validate the performance of PG-Seq™ using euploid, single and double trisomy and segmental aneuploidy single cells and 5-cells.
Innovative embryo identification using PG-Seq™
Mitochondria are maternally inherited. The mitochondrial genome (mtDNA) contains single nucleotide variants (SNVs). PG-Seq™ with DOPlify™ WGA provides superior amplification of the mitochondrial genome, with 100% of the mitochondrial sequence available from 48 sample multiplexing on a MiSeq.
Using a SNV panel, the mtDNA sequence provides a novel opportunity for DNA-based confirmation of maternal origin of an embryo biopsy and sibling embryo identification, which could be used for sample tracking within an IVF or genetic service provider laboratory.
Aim – To demonstrate the use of PG-Seq™ with RHS’ Embryo ID panel to achieve accurate and economical PGS with embryo identification using low pass NGS.
Maximising clinical NGS data: The effect of WGA & sequencing read length
The amount of Next Generation Sequencing (NGS) data generated from single or small cell number DNA samples is highly influenced by the choice of Whole Genome Amplification (WGA) kit and sequencing kit. The mitochondrial genome (mtDNA) represents an ideal model to evaluate the effect of WGA and NGS methods on sequencing results due to its small size, high copy number and the consequent read depth obtainable from low pass sequencing. The mtDNA results provide insight into genome wide effects expected with more expensive deeper sequencing of the entire human genome.
Aim – To compare the effect of two WGA and NGS methodologies on mtDNA sequence data.
Non-invasive PGS using cell free DNA from spent embryo culture media
Cell-free DNA (cfDNA) has been observed in spent embryo culture media, which creates an exciting possibility for the development of non-invasive preimplantation genetic screening (PGS). Non-invasive PGS offers several advantages. Most notably this includes removing the requirement for embryologists to have biopsy training and experience. It also provides the option to perform PGS on embryos that are not able to be biopsied due to embryo conditions such as stage of hatching or positioning of the inner cell mass. In the lead up to clinical evaluation, it is critical to confirm the ability to reliably whole genome amplify (WGA) the limited cfDNA in spent embryo culture media, and determine the concordance, accuracy and sensitivity with biopsy-based PGS.
Aim – To confirm robust, accurate and reliable whole genome amplification and subsequent chromosomal analysis of cfDNA from spent embryo culture media for non-invasive PGS.
Enrichment of a BRCA1 deletion during whole genome amplification for a novel combined PGS+PGD approach
Breast cancer risk can be increased by mutations in the BRCA1 gene. It is possible to perform preimplantation genetic diagnosis (PGD) to pre-screen IVF embryos and remove the risk factor from future generations. RHS have developed a novel method for concurrent PGD and aneuploidy screening for preimplantation genetic screening (PGS) from a single embryo biopsy.
Aim – To demonstrate combined PGD and PGS using DOPlify™ and next generation sequencing (NGS) for detection of a2bp clinically-relevant BRCA1 deletion.
A combined PGD+PGS NGS solution for β-thalassemia and HLA-A typing
β-thalassemia screening (beta-globin; HBB) represents almost 15% of PGD cases, with a further 4.5% of cases combining human leukocyte antigen HLA-typing with HBB. There are more than 200 disease-causing HBB mutations described, so screening necessitates a pan-HBB mutation detection and HLA panel approach. The ability to combine β-thalassemia and HLA Preimplantation Genetic Diagnosis (PGD) with concurrent Preimplantation Genetic Screening (PGS) in one test maximizes the screening opportunity for a single embryo biopsy. RHS have developed a whole genome amplification (WGA) with DOPlify™ and Target Sequence Enrichment (TSE) protocol using gene-specific PCR that provides a novel comprehensive PGD+PGS solution.
Aim - To demonstrate that DOPlify™ with Target Sequence Enrichment achieves sensitive, accurate and economical combined PGS and PGD using low pass NGS.
Introducing PG-Seq™, a complete NGS solution for PGS
PG-Seq™ for Pre-implantation Genetic Screening offers a novel complete, cost effective workflow. Suitable for the analysis of up to 48 embryo biopsies in a single NGS run, the workflow includes DOPlify™, library preparation reagents and data analysis software.
Prior to clinical validation, it is critical to validate the performance of PG-Seq™ using cell lines of known identity.
Aim –To assess the performance of PG-SeqTMusing euploid, single and double trisomy and segmental aneuploidy single cells and 5-cells.
Validation of a high throughput, low cost NGS PGS assay: impact of library preparation and read length on resolution
Limited Whole Genome Amplification (WGA) technologies suitable for copy number detection are commercially available. RHS DOPlify™ has been specifically developed for Next Generation Sequencing (NGS). The time and financial efficiencies offered by NGS of clinical samples is mediated by multiplexing, with the cost per sample decreasing as more samples are multiplexed. However, this can also negatively impact the test resolution and quality of results. When limited source material for genome wide evaluation is available; effective fragmentation, highly efficient adapter ligation and optimal read length is imperative to maximise the reads per sample available for interrogation.
Aim–This study evaluated the impact of library preparation kits and read length on PGS results using whole genome amplified single cell and 5-cell inputs.
Combined PGD and PGS for β-Thalassemia and HLA using Targeted Sequence Enrichment
b-Thalassemia is caused by mutations within the beta globin (HBB) gene involved in haemoglobin production, with more than 200 disease causing mutations described so far.
ESHRE data suggests that b-Thalassemia screening represents almost 15% of PGD cases, with a further 4.5% of cases combining b-Thalassemia with HLA-typing. The ability to combine PGD with PGS presents an opportunity to transfer euploid embryos free of the disease causing mutation, removing the disease from the family lineage.
RHS has developed a novel combined approach using whole genome amplification (WGA) with DOPlify™ and gene-specific PCR for Target Sequence Enrichment (TSE). This approach readily allows PGD and PGS using an economical low pass, multiplexed NGS protocol.
DOPlify™ with Target Sequence Enrichment achieves sensitive, accurate and economical combined PGS and PGD using low pass NGS
Mitochondrial genome coverage for copy number determination and detection of disease; the impact of WGA
Mutations in the mitochondrial genome (mtDNA) have been linked to diseases such as cancer, diabetes and deafness. Additionally, recent data suggests that mitochondrial genome load can impact implantation potential of euploid embryos. The selection of embryos for IVF transfer using the additional information from mitochondria requires an accurate and high coverage whole genome amplification (WGA) methodology. Additionally, since the mtDNA genome is 16,571bp in length and there are multiple copies per cell, this provides a model to evaluate performance of WGA technologies.
Aim – This study aimed to compare two different commercially available WGA kits; PicoPlex® (Rubicon Genomics) and DOPlify™ (RHS Ltd), evaluating overall mtDNA genome coverage along with coverage of 23 common mitochondrial mutations using NGS of the whole genome amplified single cells.
Development of a 5 hour PGS protocol for a day 5 fresh transfer
Chromosomal aneuploidies are the main cause of abnormal development of embryos and implantation failures. Preimplantation genetic screening (PGS) allows the selection of embryos with euploid chromosomal content and increases IVF treatment efficacy. PGS microarrays are traditionally hybridised for a minimum of 3 hours to overnight. The duration of hybridisation impacts on signal intensity, with shorter times typically reducing the array signal.
Aim – This study aimed to develop a novel hybridisation solution which could significantly decrease protocol duration, enabling same workday results and providing an opportunity for routine fresh transfers of PGS screened embryos.
Validation of EmbryoCellect® with SurePlex amplified embryo biopsies
Preimplantation genetic screening (PGS) array technologies are efficient and also very practical to perform without the need to batch large numbers of samples. The RHS’ EmbryoCellect® kit has previously been validated using euploid and commercially available aneuploid single cell and multi-cell samples for whole chromosome aneuploidy screening.
Aim – To determine the cross-compatibility of SurePlex amplified embryo biopsies and RHS’ EmbryoCellect® labelling and microarray hybridisation protocols for PGS.
Next Generation Sequencing (NGS) metrics following DOP-PCR whole genome amplification (WGA) of single and multi-cell samples for PGS
Whole genome amplification (WGA) is often used to generate sufficient DNA for downstream analysis.
The aim of this study was to compare Next Generation Sequencing (NGS) workflows and aligned read data metrics from a range of NGS platforms as models for Pre-implantation Genetic Screening (PGS) and Pre-implantation Genetic Diagnosis (PGD). Comparisons were made using single cell and 5-cell aliquots amplified utilizing the Reproductive Health Science Ltd proprietary DOP-PCR based WGA as described in the DOPlifyTM and EmbryoCellectTM kits.
Combined PGD and PGS: Enrichment of PGD genes during whole genome amplification
The aim of this study was to determine the feasibility of synchronous whole genome amplification and gene specific amplification by PCR for combined PGD for monogenic disorders and PGS for aneuploidy utilizing aCGH or NGS.
Combined PGD/PGS Using Whole Genome Sequencing in a Model of Blastomer and Trophectoderm Biopsy
The aim of this study was to investigate the potential for combined preimplantation genetic diagnosis and screening of inherited and de novo mutations using whole genome sequencing.