“Can nanopore long-read sequencing replace current cytogenetic methods in clinical genetic diagnostics” was the title of Emilie Boye Lester’s session at the Nanopore Community Meeting 2022. Lester is from Odense University Hospital in Denmark and explained that the aim of their study was to “explore the capability of long-read whole genome sequencing to detect structural variants and fine map the involved breakpoints.” They study used a PromethION 24 and R9.4.1, R10.4, and R10.4.1 flow cells with the Ligation Sequencing Kit. They used the Monarch HMW DNA kit for Cells & Blood and prepared the DNA with the Circulomics Nanobind CBB kit. This is the kit I termed “the jello prep” because I could not get bacterial genomic DNA to dissolve from those preps! Lester shared a table with rows for eight samples sequenced with different flow cells at a depth of 16-30x and numerous structural variants. Graphing the sequencing data, Lester noted that there was a clear improvement when changing from R9 to R10 flow cells for the last eleven samples. For their 10.4.1 flow cells, the mean quality score was 20.1, with v 14 and 400 bps speed. For analysis, Lester and team used Super Accurate Base calling and alignment with winnowmap and minimap2. They analyzed a sample with a known translocation involving chromosomes 1 and 18. They visualized the reads using the Integrative Genomics Viewer (IGV) and noted reads and the breakpoint in the SMAD4 gene. In a second case, there was balanced and unbalanced translocation between chromosomes 4 and 18 as determined by the long-read mapping. They were able to note the deletion in chromosome 18. Lester and team concluded that Nanopore long-read sequencing for the detection of structural variants and fine mapping of chromosomal breakpoints has great potential. They were able to detect “reciprocal translocations and fine mapping of breakpoints” successfully. The team is also working to optimize a translocation tool to filter normal variation. I imagine that there is a lot more optimizing and quality control to validate this method, but it seems really useful and has great potential.
