Tom Walsh from the University of Washington in the USA spoke at London Calling about “Discovery of deeply intronic damaging mutations with nanopore sequencing.” Walsh explained that in the 1990s, genetic testing for predisposition to breast cancer began. In the 200s, additional cancer-risk genes were discovered. Now, there are numerous panels and ten genes for breast and ovarian cancer risk. However, the risk remains unresolved in many severely affected families. Short-read sequencing has been used to explore DNA from these families to find mutational mechanisms. Regulatory variants, mobile element insertions, and deeply intronic variants are not routinely evaluated. Walsh noted that adaptive sampling and nanopore sequencing can detect all three variants. The research team uses ten barcodes per sequencing run and four samples per flow cell. Walsh has explored sequences from 240 relatives from 120 families with the goal of 20X coverage. The workflow includes filtering for rare variants with gnomAD and performing splice predictions with Splice AI and Pangolin. Patient whole blood RNA was reverse transcribed with gene-specific primers and evaluated by long-read cDNA sequencing. Walsh and team discovered variants that caused pseudo-exons. The variants the team identified led to the conclusion that there is a potential hotspot for cryptic splicing events in one genomic region. Walsh noted that adaptive sampling applied to previously unsolved families can help identify deep intronic variants. Moving forward, the team wants to run more samples and increase throughput. The team also wants to apply direct RNA sequencing. I enjoyed learning about the genetic testing the research team conducted to learn about these potential hotspots.
