Targeted next generation sequencing (NGS) is the predominant methodology for the molecular genetic diagnosis of inherited conditions. In many laboratories, NGS-identified variants are routinely validated using a different method, to minimize the risk of a false-positive diagnosis. This can be particularly important when pathogenic variants are located in complex genomic regions. In this situation, new long-read sequencing technologies have potential advantages over existing alternatives. However, practical examples of their utility for diagnostic purposes remain scant. Here, we report the use of nanopore sequencing to validate a PMS2 mutation refractory to conventional methods. In a patient who presented with colorectal cancer and loss of PMS2 immunostaining, short-read NGS of Lynch syndrome-associated genes identified the recurrent PMS2 insertion-deletion variant, c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3). Confirmation of this variant using bidirectional Sanger sequencing was impeded by an upstream intron 6 poly(T) tract. Using a locus-specific amplicon template, we undertook nanopore long-read sequencing in order to assess the diagnostic accuracy of this platform. Pairwise comparison between a curated benchmark allele (derived from short-read NGS and unidirectional Sanger sequencing) and the consensus nanopore dataset revealed 100% sequence identity. Our experience provides insight into the robustness and ease of deployment of “third-generation” sequencing for accurate characterisation of pathogenic variants.

靶向下一代测序 (NGS) 是遗传疾病分子遗传学诊断的主要方法。在许多实验室中，NGS 鉴定的变异通常使用不同的方法进行验证，以最大限度地降低假阳性诊断的风险。当致病变异位于复杂的基因组区域时，这一点尤其重要。在这种情况下，新的长读长测序技术比现有的替代技术具有潜在的优势。然而，它们用于诊断目的的实际例子仍然很少。在这里，我们报告了使用纳米孔测序来验证PMS2常规方法难以发生的突变。在一名患有结直肠癌且 PMS2 免疫染色缺失的患者中，Lynch 综合征相关基因的短读 NGS 确定了复发性PMS2插入删除变体，c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3)。使用双向 Sanger 测序确认该变体受到上游内含子 6 poly(T) 束的阻碍。使用基因座特异性扩增子模板，我们进行了纳米孔长读长测序，以评估该平台的诊断准确性。策划的基准等位基因（源自短读 NGS 和单向 Sanger 测序）和共识纳米孔数据集之间的成对比较显示 100% 的序列同一性。我们的经验提供了对“第三代”测序的稳健性和易于部署的洞察力，以准确表征致病性变异。