Product Sheets and Poster Presentations


Application Notes

Download DOPlify® NGS Study


Information Sheets

Download Fundamentals of Whole Genome Amplification


Poster Presentations

ASHG 2018

A novel NGS solution for combined PGT-M and PGT-A: Analysis for -thalassemia

Thalassemia, caused by mutations within genes involved in haemoglobin production, including the haemoglobin subunit beta gene (HBB), has become one of the most prevalent blood disorders around the world and is a frequent target for Preimplantation Genetic Testing for Monogenic disorders (PGT-M). ESHRE data suggests that β-Thalassemia screening represents almost 15% of PGT-M cases, with a further 4.5% of cases combining β-Thalassemia with HLA-typing. The ability to combine aneuploidy detection (PGT-A) and PGT-M, by adding gene-specific PCR primers to a Whole Genome Amplification (WGA), maximises the screening opportunity for asingle embryobiopsy.

Aim – To demonstrate the application of a novel Target Sequence Enrichment (TSE) protocol using the DOPlify® kit for combined PGT-M for β-thalassemia and PGT-A by Next Generation Sequencing (NGS).

Download poster here


A novel single tube approach for combined PGT-A and PGT-M using Ion Torrent™ NGS

There are several Whole Genome Amplification (WGA) and library preparat ion methods to prepare embryo biopsies for Next Generation Sequencing (NGS) for Preimplantation Genetic Testing for Aneuploidy (PGT-A). The DOPlify® kit provides a flexible technology to not only amplify whole genomes but also target sequences using the RHS Target Sequence Enrichment (TSE) protocol. The attributes of the DOPlify® kit 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 low template samples for combined PGT-A and PGT-M for β-thalassemia mutation testing in a single tube for Ion Torrent™ NGS.

Download poster here


Embryo sample tracking and identification during PGT 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 the PG-Seq™ kit readily allows SNV analysis, even from a 48 sample NGS run. This information could be used for sample tracking within an IVF or genetic service provider laboratory.

Aim– To demonstrate the use of the Embryo ID panel to achieve accurate and economical embryo identification as part of routine PGT-A using the PG-Seq™ kit.

Download poster here


ASRM 2018

Accurate combined preimplantation genetic testing with DOPlify® WGA and target sequence enrichment

Allele Drop Out (ADO), or failed PCR amplification of one or both alleles, presents a significant risk of misdiagnosis or the return of a “noresult” for Preimplantation Genetic Testing for monogenic disease (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 an euploidy 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 PGT-M is an advantage of the DOPlify® kit and Target Sequence Enrichment (TSE) protocol (patent pending).

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.

Download poster here


Embryo biopsy to tube transfer –How much PBS is too much?

When an embryo biopsy is performed prior to Preimplantation Genetic Testing for Aneuploidy (PGT-A), the biopsy is washed in PBS or similar buffer to remove residual culture media before being placed in a tube for Whole Genome Amplification (WGA). The amount of buffer transferred to the tube can vary from the minimal recommended amount (<2μL) to a larger volume. It is unknown what effect increased volumes of transfer buffer have on the WGA and subsequent PGT-A outcomes.

Aim – This study aimed to determine the maximum volume of PBS that can be transferred into a tube with an embryo biopsy without compromising WGA using the DOPlify® kit.

Download poster here


Tailoring resolution for PGT-A and PGT-SR by modifying NGS sample throughput 

Unlike traditional DNA analysis platforms used for Preimplantation Genetic Testing (PGT), Next Generation Sequencing (NGS) technologies provide a highly flexible and customizable workflow for the detection of segmental copy number variations (CNVs) offering variable resolution. However, greater resolution is achieved at the expense of sample throughput.

Aim – We aim to define the level of resolution achievable using the PG-Seq™ kit with different NGS sample throughput.

Download poster here


Combined β-thalassemia mutation detection and PGT-A using a novel PCR barcoding approach for Ion Torrent™ NGS

Thalassemia was the 4th most frequent monogenic disorder screened using Preimplantation Genetic Testing (PGT-M) according to 10 years of ESHRE data collection. The ability to combine β-thalassemia-specific PCR primers and Whole Genome Amplification (WGA) for concurrent PGT-M and PGT for aneuploidy (PGT-A) together in a single PCR reaction represents a unique approach to maximise the screening opportunity for a single embryobiopsy. DOPlify® WGA provides a flexible technology to amplify whole genomes and clinically relevant sequences using the Target Sequence Enrichment (TSE) protocol and also a unique mechanism to incorporate specific sequences needed for Next Generation Sequencing (NGS) sample preparation, offering a streamlined solution for combined PGT-M and PGT-A.

Aim – To develop a novel approach that allows amplification and PCR barcoding of low template samples for combined PGT-A and PGT-M for β-thalassemia mutation testing in asingle tube for Ion Torrent™ NGS.

Download poster here


Embryo sample tracking 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 (PGT-A) by Next Generation Sequencing (NGS) and the depth and breadth of coverage obtained from the PG-Seq™ kit readily allows SNV analysis, even froma 48 sample NGS run. This information could be used for sample tracking within an IVF orgenetic service provider laboratory.

Aim – To demonstrate the use of the Embryo ID panel to achieve accurate and economical embryo identification as part of routine PGT-A using the PG-Seq™ kit.

Download poster here


Strategies to achieve combined non-invasive PGT-M & PGT-A on spent culture media using target sequence enrichment

Theclinical 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 aeuploid 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 Day5-7 using DOPlify® kit (Laneetal, 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. Optimisation of a Whole Genome Amplification (WGA) reaction along with specific primers in a single PCR reaction allows both PGT-A and targeted higher resolution Nex tGeneration Sequencing (NGS) providing a strategy to combine monogenic disease detection (PGT-M) with PGT-A.

Aim  - To identify an optimal protocol to amplify DNA in spent embryo culture media that maximises WGA DNA yield and NGS results, and demonstrate the development of a combined PGT-M andPGT-A workflow for spent embryo culture media.

Download poster here


OZ Single Cell 2018

Whole genome amplification: no method is the same

Currently, single cell and low copy number DNA templates require whole genome amplification (WGA) prior to downstream DNA analysis. There are a number of commercially available kits, each with distinctability to generatere presentative coverage of the complete genome. Ideally, the amplification should be robust, scalable and automation ready for high throughput analysis. More importantly, non-biased coverage should be achieved, with a resolution compatible with single nucleotide variant (SNV) and copy number variation (CNV) detection.
There are a small number of single cell whole genome amplification kits available on themarket. It is seldom known or appreciated that the method of whole genome amplification used in these kits is not consistent and that each method has its own mechanism by which it replicates the DNA template with different amplification biases due to the processivity and fidelity of enzymes used.

Aim - The purpose of this review is to compare and contrast the 4 main whole genome amplification technologies currently available in kits, including their hands on and incubation times.

Download poster here


Combined multiplex target enrichment and CNV detection using a single NGS index

Combining Whole Genome Amplification (WGA) along with gene-specific primers in a single PCR reaction to simultaneously allow copy number variation (CNV) detection and high resolution DNA analysis from a single cell or low-DNA template sample is an advantage of the DOPlify® kit and the Target Sequence Enrichment (TSE) protocol.
Aim – The aim of this study was to demonstrate a novel multiplex sequence enrichment protocol using the DOPlify® kit for combined detection of a 2bp heterozygous deletion and CNV detection using a single sequencing index and low-pass Next Generation Sequencing (NGS).

Download poster here


ESHRE 2018

Customising the limit of detection for PGT-A and PGT-SR using NGS

Unlike traditional DNA analysis platforms used for Preimplantation Genetic Testing (PGT), Next Generation Sequencing (NGS) technologies provide a highly flexible and customisable workflow for the detection of segmental copy number variations (CNVs) offering variable resolution. However, greater resolutionis achieved at the expense of sample throughput.

Aim–We aim to define the level of resolution achievable using the PG-Seq™ kit with different NGS sample throughput.

Download poster here


Validation of using the mitochondrial genome for embryo identification

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 embryobiopsies and sibling embryo identification.The mitochondrial genome is sequenced during Preimplantation Genetic Testing for Aneuploidy (PGT-A) by Next Generation Sequencing (NGS) and the depth and breadth of coverage obtained from the PG-Seq™kit readily allows SNV analysis, even from a 48 sample NGS run. This 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 the PG-Seq™ kit.

Download poster here


PGDIS 2018

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™.

Download poster here


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.

Download poster here


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).

Download poster here


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.

Download poster here


ASRM 2017 

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.

Download WGA and NGS read length poster


FSA 2017

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.

Download PG- Seq™- an accurate complete NGS solution for PGS Poster


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.

Download Innovative embryo identification using PG-Seq™ Poster


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.

Download  Maximising clinical NGS data: The effect of WGA & sequencing read length Poster


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.

Download Non-invasive PGS using cell free DNA from spent embryo culture media Poster


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.

Download Enrichment of a BRCA1 deletion during whole genome amplification for a novel combined PGS+PGD approach Poster


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.

Download A combined PGD+PGS NGS solution for β-thalassemia and HLA-A typing Poster


Poster Presentations

ESHRE 2017

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.

Download Introducing PG-Seq™, a complete NGS solution for PGS Poster

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.

Download Validation of a high throughput, low cost NGS PGS assay: impact of library preparation and read length on resolution Poster

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

Download Combined PGD and PGS for β-Thalassemia and HLA using Targeted Sequence Enrichment Poster


PGDIS 2017

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.

Download Mitochondrial genome coverage for copy number determination and detection of disease; the impact of WGA Poster

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.

Download  Development of a 5 hour PGS protocol for a day 5 fresh transfer Poster


ESHRE 2016 

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.

Download ESHRE 2016 Poster Presentation on Next Generation Sequencing (NGS)


PGDIS 2016

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.

Download PDGIS 2016 Combines PGD and PGS : Enrichment of PGD genes during whole genome amplification Poster