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.

中文翻译：

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使用放大的多扩增子长读长测序方法对从头突变进行定相

从头突变 (DNM) 在降低健康的严重遗传疾病中起着重要作用。为了更好地了解它们在疾病中的作用，重要的是确定引起这些突变的突变事件的起源和时间，特别是在男性不育症等性别特异性发育障碍中。然而，目前可用的短读长测序方法并不非常适合定相，因为这需要跨越 DNM 和一个或多个信息单核苷酸多态性的长连续 DNA 链。为了克服这些挑战，我们使用 Oxford Nanopore Technologies MinION 平台优化并实施了多重长读长测序方法。我们专注于改进目标扩增，将长读长测序数据与高质量的短读长序列数据相结合，并开发锚定相位计算方法。这种方法解决了长程目标扩增固有的定相挑战以及与长读长测序相关的测序错误的正常累积。总共有 109 个 DNM 中的 77 个 (71%) 被成功分期并确定了来源亲本。大多数定相 DNM 是合子前的 (90%)，其准确性由 49.6% 的平均突变等位基因频率和 0.84% 的标准误差突出显示。本研究展示了采用集成的短读长和长读长测序方法进行大规模 DNM 定相的好处。109 个 DNM 中的 77 个 (71%) 已成功分期并确定了原产地。大多数定相 DNM 是合子前的 (90%)，其准确性由 49.6% 的平均突变等位基因频率和 0.84% 的标准误差突出显示。本研究展示了采用集成的短读长和长读长测序方法进行大规模 DNM 定相的好处。109 个 DNM 中的 77 个 (71%) 已成功分期并确定了原产地。大多数定相 DNM 是合子前的 (90%)，其准确性由 49.6% 的平均突变等位基因频率和 0.84% 的标准误差突出显示。本研究展示了采用集成的短读长和长读长测序方法进行大规模 DNM 定相的好处。