Phasing of de novo mutations using a scaled‐up multiple amplicon long‐read sequencing approach

Holt, Giles; Batty, Lois; Alobaidi, Bilal; Smith, Hannah; Oud, Manon S; Ramos, Liliana; Xavier, Miguel J; Veltman, Joris A.

doi:10.1002/humu.24450

Cited by 4 publications

(1 citation statement)

References 54 publications

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“…Therefore, further exploration is required to improve the detectable rate. One potential approach to achieve this is through the use of multiplexed long-read sequencing [ 36 ]. Moreover, it is hard to figure out how those DNMs were associated with the phenotype, and the absence of information about children’s diseases restricted the clinical significance of assessing those DNMs.…”

Section: Discussionmentioning

confidence: 99%

Post-implantation analysis of genomic variations in the progeny from developing fetus to birth

Zheng,

Lin,

Wang

et al. 2024

Hum Genomics

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The analysis of genomic variations in offspring after implantation has been infrequently studied. In this study, we aim to investigate the extent of de novo mutations in humans from developing fetus to birth. Using high-depth whole-genome sequencing, 443 parent-offspring trios were studied to compare the results of de novo mutations (DNMs) between different groups. The focus was on fetuses and newborns, with DNA samples obtained from the families’ blood and the aspirated embryonic tissues subjected to deep sequencing. It was observed that the average number of total DNMs in the newborns group was 56.26 (54.17–58.35), which appeared to be lower than that the multifetal reduction group, which was 76.05 (69.70–82.40) (F = 2.42, P = 0.12). However, after adjusting for parental age and maternal pre-pregnancy body mass index (BMI), significant differences were found between the two groups. The analysis was further divided into single nucleotide variants (SNVs) and insertion/deletion of a small number of bases (indels), and it was discovered that the average number of de novo SNVs associated with the multifetal reduction group and the newborn group was 49.89 (45.59–54.20) and 51.09 (49.22–52.96), respectively. No significant differences were noted between the groups (F = 1.01, P = 0.32). However, a significant difference was observed for de novo indels, with a higher average number found in the multifetal reduction group compared to the newborn group (F = 194.17, P < 0.001). The average number of de novo indels among the multifetal reduction group and the newborn group was 26.26 (23.27–29.05) and 5.17 (4.82–5.52), respectively. To conclude, it has been observed that the quantity of de novo indels in the newborns experiences a significant decrease when compared to that in the aspirated embryonic tissues (7–9 weeks). This phenomenon is evident across all genomic regions, highlighting the adverse effects of de novo indels on the fetus and emphasizing the significance of embryonic implantation and intrauterine growth in human genetic selection mechanisms.

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Section: Discussionmentioning

confidence: 99%

Post-implantation analysis of genomic variations in the progeny from developing fetus to birth

Zheng,

Lin,

Wang

et al. 2024

Hum Genomics

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Phasing of de novo mutations using a scaled‐up multiple amplicon long‐read sequencing approach

et al. 2022

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De novo mutations (DNMs) play an important role in severe genetic disorders that reduce fitness. To better understand their role in disease, it is important to determine the parent-of-origin and timing of mutational events that give rise to these mutations, especially in sex-specific developmental disorders such as male infertility. However, currently available short-read sequencing approaches are not ideally suited for phasing, as this requires long continuous DNA strands that span both the DNM and one or more informative single-nucleotide polymorphisms. To overcome these challenges, we optimized and implemented a multiplexed long-read sequencing approach using Oxford Nanopore technologies MinION platform. We focused on improving target amplification, integrating long-read sequenced data with high-quality short-read sequence data, and developing an anchored phasing computational method. This approach handled the inherent phasing challenges of long-range target amplification and the normal accumulation of sequencing error associated with long-read sequencing. In total, 77 of 109 DNMs (71%) were successfully phased and parent-of-origin identified. The majority of phased DNMs were prezygotic (90%), the accuracy of which is highlighted by an average mutant allele frequency of 49.6% and standard error of 0.84%. This study demonstrates the benefits of employing an integrated short-read and long-read sequencing approach for large-scale DNM phasing.DNM, long-read sequencing, parent-of-origin, phasing, targeted sequencing, WES | INTRODUCTIONDe novo mutations (DNMs) arise from mutational events that occur during gametogenesis in either parent germ cells, or postzygotically in both somatic and germ cells of the individual carrying them. On average, one to two DNMs can be found in the coding region of a person's genome (Durbin et al., 2010;O'roak et al., 2011;Xu et al., 2011). DNMs are of particular significance due to their contribution to many diseases and genetic disorders, notably those affecting individual fitness such as intellectual disability and male infertility

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Novel Genes of the Male Reproductive System: Potential Roles in Male Reproduction and as Non‐hormonal Male Contraceptive Targets

Garcia,

Matzuk

2024

Molecular Reproduction Devel

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The development of novel non‐hormonal male contraceptives represents a pivotal frontier in reproductive health, driven by the need for safe, effective, and reversible contraceptive methods. This comprehensive review explores the genetic underpinnings of male fertility, emphasizing the crucial roles of specific genes and structural variants (SVs) identified through advanced sequencing technologies such as long‐read sequencing (LRS). LRS has revolutionized the detection of structural variants and complex genomic regions, offering unprecedented precision and resolution over traditional next‐generation sequencing (NGS). Key genetic targets, including those implicated in spermatogenesis and sperm motility, are highlighted, showcasing their potential as non‐hormonal contraceptive targets. The review delves into the systematic identification and validation of male reproductive tract‐specific genes, utilizing advanced transcriptomics and genomics studies with validation using novel knockout mouse models. We discuss the innovative application of small molecule inhibitors, developed through platforms like DNA‐encoded chemistry technology (DEC‐Tec), which have shown significant promise in preclinical models. Notable examples include inhibitors targeting serine/threonine kinase 33 (STK33), soluble adenylyl cyclase (sAC), cyclin‐dependent kinase 2 (CDK2), and bromodomain testis associated (BRDT), each demonstrating nanomolar affinity and potential for reversible and specific inhibition of male fertility. This review also honors the contributions of Dr. David L. Garbers whose foundational work has paved the way for these advancements. The integration of genomic, proteomic, and chemical biology approaches, supported by interdisciplinary collaboration, is poised to transform male contraceptive development. Future perspectives emphasize the need for continued innovation and rigorous testing to bring these novel contraceptives from the laboratory to clinical application, promising a new era of male reproductive health management.

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Phasing of de novo mutations using a scaled‐up multiple amplicon long‐read sequencing approach

Cited by 4 publications

References 54 publications

Post-implantation analysis of genomic variations in the progeny from developing fetus to birth

Post-implantation analysis of genomic variations in the progeny from developing fetus to birth

Phasing of de novo mutations using a scaled‐up multiple amplicon long‐read sequencing approach

Novel Genes of the Male Reproductive System: Potential Roles in Male Reproduction and as Non‐hormonal Male Contraceptive Targets

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