Dan Turner from Oxford Nanopore Technologies presented at London Calling 2019 on “The real Simon Pure.” I was intrigued by the title and wanted to learn more. Turner is part of the ONT Applications Team and explained the different groups represented, including the VolTRAX2 team. The real Simon Pure title, Turner explained, is from a book. They spoke about what sequencing a genome means and how Nanopore offers unique features. In a graph, they compared native and PCR sequencing of a genome, highlighting regions of low coverage with PCR. Turner noted that using PCR-amplified libraries reduces read length and eliminates methylation information. Citing examples, PCR is affected by G+C content. Friedreich’s Ataxia, a neurodegenerative disorder, was the goal of an ONT PCR-free target enrichment. If a person has both alleles, they are affected. One allele results in carrier status. Turner and team chose to use Cas9 to enrich for the ataxia region of interest and sequence parents and children samples. In affected children, CpG methylation was assessed with Nanopolish, and the expanded haplotypes were hypermethylated. Turner also presented data from bacterial genomes. Their goal was to analyze metagenomes and use methylation for binning. The team took E. coli K-12 wild-type and dam mutant. They then used methylation to partition genomes and associate plasmids with their hosts since each strain had a different plasmid. Turner spoke about the use of Pore-C chromatin conformation capture to improve assembly. In Pore-C, a contact map is generated. Pore-C was used to compare two human cell lines, identifying rearrangements and copy number changes. Using a combination of ultra-long (100 kb+) and shorter reads with Pore-C, contiguous assemblies were produced with more accuracy. This session had several key ideas and techniques that are useful to learn about.
