STUDY QUESTION Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? SUMMARY ANSWER Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. WHAT IS KNOWN ALREADY Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. STUDY DESIGN, SIZE, DURATION This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5–10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. MAIN RESULTS AND THE ROLE OF CHANCE The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. LIMITATIONS, REASONS FOR CAUTION The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. WIDER IMPLICATIONS OF THE FINDINGS Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 ‘Methods for haplotyping single-cells’ and ZL913096-PCT/EP2014/068315-WO/2015/028576 ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’ licensed to Agilent Technologies. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

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Study question Can we use comprehensive genome-wide haplotyping PGT for couples carrying de novo mutations, given the absence of phasing references to establish the disease-associated haplotype? Summary answer Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. What is known already Preimplantation genetic testing (PGT) aims to select embryos devoid of inherited pathogenic variants. Current comprehensive genome-wide haplotyping PGT methods cannot be applied for couples where one of the partners carries a de novo mutation, as absence of affected close relatives restricts variant phasing to establish the disease-associated haplotype. For such families targeted approaches are currently used instead, thus missing genome-wide embryo analysis. Study design, size, duration To overcome the forementioned limitation, we developed a long-read amplicon guided haplotype imputation method. From January 2017 till June 2021 thirty-two couples were enrolled into the PGT program at the Centre for Human Genetics, UZ Leuven, in the context of a de novo variant causing a known monogenic disorder in one of the two partners. Participants/materials, setting, methods Genomic DNA from the partner carrying the variant (proband) and his/her parents was used for trio analysis via long-range PCR and long-read amplicon sequencing using the PacBio RSII and/or Oxford Nanopore platforms. Targeted haplotype phasing was then performed to impute the disease-associated allele. Main results and the role of chance The parental origin of the mutant allele was identified in 20 patients, resulting in the current 62.5% success rate. Most de novo mutations occurred on the paternal allele. In the remaining families, the region of interest either had no SNPs or had insufficient number of informative SNPs linked to mutation. From the successfully analyzed couples, eight have proceeded to PGT cycle and so far, three disease-free children have been born. Limitations, reasons for caution A costly family-specific work-up is required, allowing nevertheless the implementation of a generic genome-wide method at the embryo analysis stage. The sequencing cost itself is anticipated to decrease in time. To avoid non-informative results, the analysis of multiple amlpicons is recommended. Wider implications of the findings Current practices for the handling of couples with de novo variants are mostly targeted, require the combination of direct and indirect approaches and often require the multiple biopsies. Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. Trial registration number not applicable

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