Embryo tracking system for high-throughput sequencing-based preimplantation genetic testing

Abstract

STUDY QUESTION Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)? SUMMARY ANSWER Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially. WHAT IS KNOWN ALREADY Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach. STUDY DESIGN, SIZE, DURATION We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS. PARTICIPANTS/MATERIALS, SETTING, METHODS We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined. MAIN RESULTS AND THE ROLE OF CHANCE Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods. LIMITATIONS, REASONS FOR CAUTION Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT. WIDER IMPLICATIONS OF THE FINDINGS The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing. STUDY FUNDING/COMPETING INTEREST(S) M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. TRIAL REGISTRATION NUMBER N/A.

Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

“…GBS and WGS data were analysed using a modified version of the siCHILD analytical pipeline that is equipped with haplarithmisis 68 and has been further adapted for sequencing data 63 . Initially, a preparatory test was conducted for the parents and close relative(s) to assess whether the couple was eligible for PGT.…”

“…Whole genome sequencing sample processing DNA from parents and close relative(s) and WGAed DNA from embryos, (see above) that was stored according to clinical standards, was subjected to whole genome sequencing (WGS) library preparation. Briefly, a minimum input of 20 μl, with a concentration of 30 ng/μl, was supplemented with 0.12 ng of embryo tracking system (ETS) fragments (concentration: 0.03 ng/μl) 63 . Subsequently, bead-linked transposome (BLT) PCR-free library preparation (Illumina, San Diego) was carried out according to the manufacturer's instructions for input quantities ranging from 300 to 2000 ng.…”

“…Using this pipeline, aligned sequencing data was processed using Joint HaplotypeCaller from GATK (v. 3.4-46) 69 to extract the genomic locations in the dbSNP database (v. 150). All subsequent processing was carried out in R (version 3.3.1) 70 as previously described 63,68 . Briefly, the GATK output was used to determine the genotype per position of each sample using vcfR (v. 1.8.0.9000) 71 R package.…”