Long-read nanopore sequencing enables accurate confirmation of a recurrent PMS2 insertion-deletion variant located in a region of complex genomic architecture

Cancer Genet. 2021 Aug:256-257:122-126. doi: 10.1016/j.cancergen.2021.05.012. Epub 2021 May 28.


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.

Keywords: Nanopore sequencing; PMS2; Variant validation.

Publication types

  • Case Reports

MeSH terms

  • Adult
  • Female
  • Genome, Human*
  • Humans
  • INDEL Mutation / genetics*
  • Male
  • Mismatch Repair Endonuclease PMS2 / genetics*
  • Nanopore Sequencing*
  • Pedigree


  • PMS2 protein, human
  • Mismatch Repair Endonuclease PMS2