Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing

Abstract

STUDY QUESTION Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? SUMMARY ANSWER Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. WHAT IS KNOWN ALREADY Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. STUDY DESIGN, SIZE, DURATION This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. PARTICIPANTS/MATERIALS, SETTING, METHODS Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. MAIN RESULTS AND THE ROLE OF CHANCE PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). LIMITATIONS, REASONS FOR CAUTION Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. WIDER IMPLICATIONS OF THE FINDINGS Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. STUDY FUNDING/COMPETING INTEREST(S) Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 ‘Methods for haplotyping single cells’ and ZL913096-PCT/EP2014/068315 ‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 ‘High-throughput genotyping by sequencing’. Haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’) has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

Preimplantation Genetic Testing for Monogenic Disorders

“…The OnePGT solution, commercialized by Agilent, is a NGS-based generic application for PGT-M, PGT-SR, and PGT-A [8]. The method takes advantage of reduced-representation genome sequencing in offering a single workflow, starting from MDA-based WGA and followed by library preparation with double restriction enzyme digestion and enrichment for fragments in a specific range of length; sequencing data interpretation relies on the haplarithmisis algorithm with concurrent chromosome copy number detection and SNP linkage-based haplotyping.…”

“…Biopsy at the blastocyst stage followed by genome-wide technologies began to replace this gold-standard method over the last decade. The genome-wide methods yield data on genotyping as well as on chromosome copy number, allowing for concurrent analysis of PGT-M and PGT-A [4][5][6][7][8].…”

PREIMPLANTATION GENETIC TESTING: Clinical experience of preimplantation genetic testing

“…WGA followed by genome-wide methods based on SNP array or sequencing, represents a truly comprehensive approach. The current SNP array or sequencingbased approaches for PGT-M share the same principle of linkage-based testing as multiplex PCR, but the generic workflows are much more standardised and uniform, without the need for preclinical workups (Handyside et al 2010, Masset et al 2019. The major requirements for haplotyping are that the monogenic or the chromosomal aberration(s) is/are inherited and that relevant family samples are available.…”

PREIMPLANTATION GENETIC TESTING: Single-cell technologies at the forefront of PGT and embryo research

“…Alternatively, genotyping-by-sequencing (GBS) can be used to reduce genome size and complexity for cost-effective SNP genotyping (Elshire et al 2011). This principle was recently integrated into the haplarithmisis workflow for genome-wide haplotyping and copy number profiling (Zamani Esteki et al 2019a), and was also developed by Agilent Technologies as OnePGT (Masset et al 2019).…”

“…In turn, this paves the way to comprehensive PGT, which allows to combine PGT-M and PGT-SR/PGT-A to obtain information on monogenic disorders, chromosomal structural rearrangements and aneuploidy from a single biopsy using one single assay ( Fig. 1) (Natesan et al 2014, Konstantinidis et al 2015, Ben-Nagi et al 2017, Dimitriadou et al 2017, Masset et al 2019. The main difference between the two approaches is that karyomapping uses only discrete SNP genotypes, which for instance allows to identify meiotic trisomies following haplotyping, while haplarithmisis can also identify postzygotic mitotic trisomies by using haplotyped SNP B-allele frequency values.…”