Today was Aurelio’s fifth birthday! Tonight, I watched Timothy Gilpatrick’s London Calling 2019 session on “Targeted nanopore sequencing with Cas9 for studies of methylation structural variants and mutations.” Gilpatrick is a Johns Hopkins University. They are interested in specific loci and want to generate high coverage of those areas to examine structural variation. The enrichment method consists of dephosphorylating ends and then cutting with Cas9. The unique 5′ phosphate sites are then used to ligate sequencing adaptors. The example Gilpatrick provided was of an 18 kb region they targeted with two Cas9 cuts. With this method, they designed a panel of guide RNAs to target ten regions. With 3 ug of gDNA and a MinION or even a Flongle flow cell, they were able to generate enough coverage to examine these regions. With the MinION flow cell, they obtained ~80,000 reads with 1,390 on target. With the Flongle, they obtained 12,900 reads and 462 on target. Gilpatrick then used a breast cancer cell line to target an 8 kb deletion on chromosome 7. The team used Sniffles to call structural variants. They could then assign reads to the parental allele. Gilpatrick did mention that they think they are losing some long reads during the bead purification steps. They also used the Nanopolish algorithm to detect variants and use the electrical signal with increased sensitivity. The team noticed false positives and began filtering by read direction. Gilpatrick then spoke about the use of this method to interrogate the methylation state. With Isac Lee, they developed a method to convert nanopore reads using Nanopolish methylation calls and visualize methylation. Next, Gilpatrick wants to tile guide RNAs to evaluate structural variants over larger regions of interest. Oxford Nanopore Technologies no longer sells the original Cas9 kits, yet I imagine the community has developed new adaptations.
