Oral Presentation 46th Lorne Genome Conference 2025

The use of long-read sequencing to improve diagnosis of rare inherited disease (114788)

Meutia Kumaheri 1 2 , Tonia Russell 1 , Melissa Rapadas 1 , Jillian Hammond 1 , Igor Stevanovski 1 , Leah Kemp 1 , Sasha Jenner 1 , Katrina Bell 3 , Cas Simons 4 5 , Amali Mallawaarachchi 1 6 , Owen Siggs 1 , Elizabeth Palmer 7 , Gina Natoli 8 9 , Tiong Yang Tan 10 , Daniel MacArthur 4 5 , Sue White 3 , Hasindu Gamaarachchi 1 11 , Andre Martin Reis 1 2 , Ira Deveson 1 2
  1. Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  2. St Vincent Clinical School, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia
  3. Murdoch's Children Research Institute, Melbourne, Victoria, Australia
  4. Centre for Population Genomics, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  5. Centre for Population Genomics, Murdoch's Children Research Institute, Melbourne, Victoria, Australia
  6. Royal Prince Alfred Hospital, Sydney, New South Wales, Australia
  7. Sydney Children’s Hospital Network, Sydney, New South Wales, Australia
  8. Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia
  9. University of Western Australia, Perth, Western Australia, Australia
  10. Victorian Clinical Genetics Services, Melbourne, Victoria, Australia
  11. Faculty of Computer Science, University of New South Wales, New South Wales, Australia

Over half of rare inherited disease cases remain undiagnosed after exome or genome analysis with short-read sequencing (SRS), likely due to structural variants or complex repetitive regions that SRS cannot resolve. Long-read sequencing (LRS) offers improved detection of structural variants, better accuracy in repetitive regions, enhanced genome assembly and phasing, direct epigenetic modification detection, and flexible, cost-effective targeted sequencing. We aim to use LRS in undiagnosed participants from our collaborative network, including rare disease programs like RDNow, UDN-Aus, and GeneAdd, and evaluate its impact in diagnostic rates and the ability to streamline analysis. We triage participants based on parental availability (complete vs. incomplete trios) and prior analysis (half-solved, last resort or first line). For cases with suspected candidate genes or variants, we perform targeted nanopore sequencing; for cases without leads and first-line cases, we use PacBio whole-genome sequencing. Samples are then processed through our custom pipeline, Pipeface, for alignment, variant calling, and annotation. Variants are curated and prioritised in our Shiny app, JigSeq PuzzleApp. To date, we have recruited and sequenced 36 first line, 30 half-solved, and 53 last resort cases, with 111 complete trios and 18 incomplete. We have analysed 19 cases and solved 5 cases. Most solved cases involved phasing candidate variants in incomplete trios, but we are now focusing on SV analysis in first line trios. We have established an efficient system for processing and analysing undiagnosed cases, particularly effective for half-solved and incomplete trios. To further improve diagnostic rates in first line and last resort cases, we are focusing on enhancing our pipeline, specifically in SV annotation and STR genotyping. In summary, LRS has already proven valuable in resolving complex cases refractory to traditional SRS. By refining our pipeline, we aim to further boost diagnostic rates, establishing LRS as a key tool in rare disease diagnostics.