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A novel single tube amplification and barcoding approach for preimplantation genetic testing (PGT-A) for aneuploidy detectionusing Next Generation Sequencing
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RBMO VOLUME 38 ISSUE S1 2019
FIGURE 1-PERCENTAGES OF EUPLOID, ANEUPLOID AND WHOLE CHROMOSOMAL MOSAIC EMBRYOS BY AGE GROUPS.
couple the SNP is present in the paternal mutant allele and maternal wild-type allele. The results from SNP analysis were then compared with the genotype results obtained from direct sequencing on the targeted mutation, and the comparison showed 100% concordance. Aneuploidy screening on unaffected and heterozygous carrier blastocyst revealed 6 euploid, 7 mosaic and 5 aneuploid blastocysts. Five blastocysts (3 unaffected euploid, 1 heterozygous carrier euploid and 1 heterozygous carrier mosaic) were transferred with three successful implantations (2 unaffected euploid and 1 heterozygous carrier mosaic). Conclusion: The presence of wild-type allele means that the blastocyst is either unaffected or is a heterozygous carrier and vice-versa. On the other hand, the presence of mutant allele means that the blastocyst is either affected or is a heterozygous carrier and vice-versa. This in-house developed SNP assay shows concordance results with the established mutation screening assay. The combination of SNP and mutation screening assay improves the accuracy of PGD by minimizing the risk of misdiagnosis caused by allele drop out. doi: 10.1016/j.rbmo.2019.03.080
A NOVEL SINGLE TUBE AMPLIFICATION AND BARCODING APPROACH FOR PREIMPLANTATION
GENETIC TESTING (PGT-A) FOR ANEUPLOIDY DETECTIONUSING NEXT GENERATION SEQUENCING
Melinda Jasper, Steven Myers Introduction: There are several methods to prepare embryo biopsy samples for Next Generation Sequencing (NGS) for Preimplantation Genetic Testing for Aneuploidy (PGT-A). DOPlify® provides a flexible technology to not only amplify whole genomes and target sequences using RHS’ target sequence enrichment (TSE) protocol, but also provides a mechanism to incorporate platform specific sequences for NGS. The use of PCR barcoding during whole genome amplification provides several laboratory efficiencies compared to sequential whole genome amplification followed by library preparation methods, including the reduction of hands on time, total protocol time and reagent requirements for sample preparation, all of which are equally relevant across small clinics through to high throughput service laboratories. Here we describe the development of a novel approach which allows PCR barcoding of PGT-A samples in a single tube using PCR that has been developed on multi- cell samples of known ploidy as a model of trophectoderm biopsy. Material and Methods: Five-cell samples manually sorted from aneuploid cell lines (Coriell Institute)
were prepared for sequencing using a two-stage, single tube protocol. DNA was amplified using standard DOPlify® kit reagents (RHS Ltd) then Ion Torrent (ThermoFisher) NGS adapter sequences and barcodes were subsequently incorporated utilising a second PCR step within the same tube. Incorporation of the adapter sequences at both the 5’ and 3’ ends of the amplified DNA was quantified using qPCR (Kapa Biosystems) with adapter sequencespecific primers (RHS Ltd). The barcoded samples were pooled and sequenced. The sequencing data was bioinformatically aligned to hg19, sequencing metrics collated and the data analysed to determine sample ploidy status. Results: More than 8 different barcoding methods were trialled and 3 were successful. Measurable differences were observed for WGA DNA yield, DNA fragment size range and the 5’ and 3’ adapter sequence incorporation ratio (1:1-1:5). For the successful protocols, concordance of the sample karyotype was achieved for all cell lines sequenced (n=5 for each method). The most time efficient protocol produced amplified, sequencing ready samples within a total time of 3 hrs 30 min with a hands on time of about 30 mins, with further reduction of total protocol time to less than 3 hrs currently being evaluated. Additional time improvements may come from automation.
RBMO VOLUME 38 ISSUE S1 2019
80 60 40 20 0 Type A
Type B
Implantation (%)
Type C LBR (%)
TABLE 1.
Conclusions: Leveraging the unique DOPlify® technology, this novel method provides a single tube amplification and barcoding protocol to allow rapid, scalable and economical sequencing of embryo biopsy samples for PGT-A using next generation sequencing. With the possibility of incorporating RHS’ target sequence enrichment (TSE) protocol, this novel PCR barcoding approach will then allow combined PGT-M and PGT-A. Keywords: Barcoding, Indexing, NGS, PGT-A, WGA
doi: 10.1016/j.rbmo.2019.03.081
35. BIOETHICS OF PREIMPLANTATION GENETIC TESTING IN BAHRAIN AND THE GULF COOPERATION COUNCIL (GCC) COUNTRIES
Maryam Dashti, Bs.c., Dipl. Biol., M.Phil., Ph.D. Department of Molecular Medicine, College of Medicine and Medical Sciences, Arabian Gulf University, Kingdome of Bahrain
Introduction: Pre- implantation Genetic Testing (PGT), including Pre- Implantation Genetic Screening (PGS) and Pre-Implantation Genetic Diagnosis (PGD) is defined as a group of genetic analysis applied to embryos produced through IVF programs. In Bahrain, these investigations were introduced since 2007, by referring embryo biopsy samples to PGD labs outside the country. Most of the tests were initially implemented using the techniques of FISH and PCR, while a-CGH and NGS emerged later.
Genetic testing of embryos is laborious, costly, stressful and psychologically harsh on families. It should be organized and protected to ensure safety of testing and authenticity of outcome. Currently, the PGT practices in Bahrain and some of GCC countries lack many elements of quality management and require strategies for transformation of commercially oriented business to a patient-welfare oriented service. This study will present PGT data collected in the last 10 years from three major IVF centres in Bahrain. It will basically emphasize on the problems and the weaknesses encountered in our system through analysis of the data and treatment outcome in terms of pregnancy success rates. Also, in an endeavour to improve the quality of PGT services and to overcome some of the implementation-related issues observed, bioethical standards helping in improvement of the system and regulation of PGT practices will be proposed and discussed. Materials and Methods: This study will present the findings of 500 infertility/PGT cases treated in three major IVF centres in Bahrain from 2007 until present. All the centres refer their samples to satellite PGD laboratories. The tests were initially focussed on aneuploidy screening for 5 chromosomes by the technique of FISH, which was gradually shifted to 24-chromosome screening by A-CGH in 2013 through 2015 and later substituted with NGS when the technique was made available. The number of cases, the types of PGT tests performed, justifications of testing and the pregnancy outcome are all reviewed, analysed and evaluated. Results: At least 70% of cases exhibited an unjustified FISH test, while 30% requested gender selection in favour of male embryos.
e51
As for screening and diagnosis of monogenic disorders, about 70% of cases displayed a trait status for hemoglobinopathies which are common in Bahrain (5%) and the eastern province of KSA (4.5%). Lower pregnancy success rates were observed mainly in FISH cases (0-15%), but they showed higher values (17- 48%) in A- CGH (p< 0.01) and in NGS cases (p< 0.001). Conclusions and Recommendations: Our health care system in the GCC needs urgent implementation of quality standards to improve the practices of pre- implantation genetic testing. In view of the current bioethical situation of genetic testing in GCC, measures for regulating practises of PGT under a strict controlling system will be suggested for future implementation. Ethical problems in genetic testing of embryoscan be avoided in GCC by considering the following: - These tests are extremely complex, laborious and costly. Therefore, the labs performing them should be subjected to strict regulations to ensure the accuracy and reliability of test results they generate. - The clinics offering genetic testing of embryos should be certified as competent and consider counselling as a mandatory part of PGT practice (part of current IVF law). - Children produced through PGT must be registered and followed up to ensure their health and welfare. - Effect of X-linked diseases on marital status of women needs to be minimized through education. - The potential impact of PGT on discrimination against children born with disabilities should be addressed. - Financial support from the public health sector for less privileged families to be encouraged. Keywords: Bioethics, GCC, PGT, FISH, PCR, A-CGH, NGS
RBMO VOLUME 38 ISSUE S1 2019
FIGURE 1-PERCENTAGES OF EUPLOID, ANEUPLOID AND WHOLE CHROMOSOMAL MOSAIC EMBRYOS BY AGE GROUPS.
couple the SNP is present in the paternal mutant allele and maternal wild-type allele. The results from SNP analysis were then compared with the genotype results obtained from direct sequencing on the targeted mutation, and the comparison showed 100% concordance. Aneuploidy screening on unaffected and heterozygous carrier blastocyst revealed 6 euploid, 7 mosaic and 5 aneuploid blastocysts. Five blastocysts (3 unaffected euploid, 1 heterozygous carrier euploid and 1 heterozygous carrier mosaic) were transferred with three successful implantations (2 unaffected euploid and 1 heterozygous carrier mosaic). Conclusion: The presence of wild-type allele means that the blastocyst is either unaffected or is a heterozygous carrier and vice-versa. On the other hand, the presence of mutant allele means that the blastocyst is either affected or is a heterozygous carrier and vice-versa. This in-house developed SNP assay shows concordance results with the established mutation screening assay. The combination of SNP and mutation screening assay improves the accuracy of PGD by minimizing the risk of misdiagnosis caused by allele drop out. doi: 10.1016/j.rbmo.2019.03.080
A NOVEL SINGLE TUBE AMPLIFICATION AND BARCODING APPROACH FOR PREIMPLANTATION
GENETIC TESTING (PGT-A) FOR ANEUPLOIDY DETECTIONUSING NEXT GENERATION SEQUENCING
Melinda Jasper, Steven Myers Introduction: There are several methods to prepare embryo biopsy samples for Next Generation Sequencing (NGS) for Preimplantation Genetic Testing for Aneuploidy (PGT-A). DOPlify® provides a flexible technology to not only amplify whole genomes and target sequences using RHS’ target sequence enrichment (TSE) protocol, but also provides a mechanism to incorporate platform specific sequences for NGS. The use of PCR barcoding during whole genome amplification provides several laboratory efficiencies compared to sequential whole genome amplification followed by library preparation methods, including the reduction of hands on time, total protocol time and reagent requirements for sample preparation, all of which are equally relevant across small clinics through to high throughput service laboratories. Here we describe the development of a novel approach which allows PCR barcoding of PGT-A samples in a single tube using PCR that has been developed on multi- cell samples of known ploidy as a model of trophectoderm biopsy. Material and Methods: Five-cell samples manually sorted from aneuploid cell lines (Coriell Institute)
were prepared for sequencing using a two-stage, single tube protocol. DNA was amplified using standard DOPlify® kit reagents (RHS Ltd) then Ion Torrent (ThermoFisher) NGS adapter sequences and barcodes were subsequently incorporated utilising a second PCR step within the same tube. Incorporation of the adapter sequences at both the 5’ and 3’ ends of the amplified DNA was quantified using qPCR (Kapa Biosystems) with adapter sequencespecific primers (RHS Ltd). The barcoded samples were pooled and sequenced. The sequencing data was bioinformatically aligned to hg19, sequencing metrics collated and the data analysed to determine sample ploidy status. Results: More than 8 different barcoding methods were trialled and 3 were successful. Measurable differences were observed for WGA DNA yield, DNA fragment size range and the 5’ and 3’ adapter sequence incorporation ratio (1:1-1:5). For the successful protocols, concordance of the sample karyotype was achieved for all cell lines sequenced (n=5 for each method). The most time efficient protocol produced amplified, sequencing ready samples within a total time of 3 hrs 30 min with a hands on time of about 30 mins, with further reduction of total protocol time to less than 3 hrs currently being evaluated. Additional time improvements may come from automation.
RBMO VOLUME 38 ISSUE S1 2019
80 60 40 20 0 Type A
Type B
Implantation (%)
Type C LBR (%)
TABLE 1.
Conclusions: Leveraging the unique DOPlify® technology, this novel method provides a single tube amplification and barcoding protocol to allow rapid, scalable and economical sequencing of embryo biopsy samples for PGT-A using next generation sequencing. With the possibility of incorporating RHS’ target sequence enrichment (TSE) protocol, this novel PCR barcoding approach will then allow combined PGT-M and PGT-A. Keywords: Barcoding, Indexing, NGS, PGT-A, WGA
doi: 10.1016/j.rbmo.2019.03.081
35. BIOETHICS OF PREIMPLANTATION GENETIC TESTING IN BAHRAIN AND THE GULF COOPERATION COUNCIL (GCC) COUNTRIES
Maryam Dashti, Bs.c., Dipl. Biol., M.Phil., Ph.D. Department of Molecular Medicine, College of Medicine and Medical Sciences, Arabian Gulf University, Kingdome of Bahrain
Introduction: Pre- implantation Genetic Testing (PGT), including Pre- Implantation Genetic Screening (PGS) and Pre-Implantation Genetic Diagnosis (PGD) is defined as a group of genetic analysis applied to embryos produced through IVF programs. In Bahrain, these investigations were introduced since 2007, by referring embryo biopsy samples to PGD labs outside the country. Most of the tests were initially implemented using the techniques of FISH and PCR, while a-CGH and NGS emerged later.
Genetic testing of embryos is laborious, costly, stressful and psychologically harsh on families. It should be organized and protected to ensure safety of testing and authenticity of outcome. Currently, the PGT practices in Bahrain and some of GCC countries lack many elements of quality management and require strategies for transformation of commercially oriented business to a patient-welfare oriented service. This study will present PGT data collected in the last 10 years from three major IVF centres in Bahrain. It will basically emphasize on the problems and the weaknesses encountered in our system through analysis of the data and treatment outcome in terms of pregnancy success rates. Also, in an endeavour to improve the quality of PGT services and to overcome some of the implementation-related issues observed, bioethical standards helping in improvement of the system and regulation of PGT practices will be proposed and discussed. Materials and Methods: This study will present the findings of 500 infertility/PGT cases treated in three major IVF centres in Bahrain from 2007 until present. All the centres refer their samples to satellite PGD laboratories. The tests were initially focussed on aneuploidy screening for 5 chromosomes by the technique of FISH, which was gradually shifted to 24-chromosome screening by A-CGH in 2013 through 2015 and later substituted with NGS when the technique was made available. The number of cases, the types of PGT tests performed, justifications of testing and the pregnancy outcome are all reviewed, analysed and evaluated. Results: At least 70% of cases exhibited an unjustified FISH test, while 30% requested gender selection in favour of male embryos.
e51
As for screening and diagnosis of monogenic disorders, about 70% of cases displayed a trait status for hemoglobinopathies which are common in Bahrain (5%) and the eastern province of KSA (4.5%). Lower pregnancy success rates were observed mainly in FISH cases (0-15%), but they showed higher values (17- 48%) in A- CGH (p< 0.01) and in NGS cases (p< 0.001). Conclusions and Recommendations: Our health care system in the GCC needs urgent implementation of quality standards to improve the practices of pre- implantation genetic testing. In view of the current bioethical situation of genetic testing in GCC, measures for regulating practises of PGT under a strict controlling system will be suggested for future implementation. Ethical problems in genetic testing of embryoscan be avoided in GCC by considering the following: - These tests are extremely complex, laborious and costly. Therefore, the labs performing them should be subjected to strict regulations to ensure the accuracy and reliability of test results they generate. - The clinics offering genetic testing of embryos should be certified as competent and consider counselling as a mandatory part of PGT practice (part of current IVF law). - Children produced through PGT must be registered and followed up to ensure their health and welfare. - Effect of X-linked diseases on marital status of women needs to be minimized through education. - The potential impact of PGT on discrimination against children born with disabilities should be addressed. - Financial support from the public health sector for less privileged families to be encouraged. Keywords: Bioethics, GCC, PGT, FISH, PCR, A-CGH, NGS