Tonight, I watched the London Calling 2019 session “Ultra-long reads and ultra-long duplications: deciphering the mysteries of the Bordetella pertussis genome.” Natalie Ring from the University of Bath in the UK was the presenter. They spoke about whooping cough and how the introduction of vaccination in the 1950s reduced the number of reported cases. In the 1990s, the acellular vaccine was introduced. However, the number of cases has increased. This may be due to increased awareness/diagnosis, vaccine efficacy, or genetic changes. Ring’s doctoral research focuses on genetic variation at the level of the whole genome. Bordetella pertussis has a highly repetitive genome. Ring used the MinION Mk1B and MIN 106 flow cells. They prepared DNA with the Ligation Sequencing Kit and the Native Barcoding Expansion Kit. Basecalling was performed using Albacore, demultiplexed with Porechop, corrected with Canu, and assembled with Unicycler. They sequenced strains from a 2012 UK whooping cough outbreak. Four of the five strains were resolved into single contigs. In the five strains, there are genome rearrangements. UK48, the strain that wasn’t resolved with Nanopore sequencing, even with ~8kb reads. Ring noticed that the genome of UK48 is larger and has more repetitive regions. There is a region of the UK48 that has enhanced coverage, similar to UK76. They believe this is due to duplications. The research team reviewed hundreds of genomes and identified duplications and deletions. Interestingly, five UK outbreak strains have this duplication… or multiple copies! To prove these genomes had duplications, they needed to perform ultra-long sequencing of these strains. They performed phenol: chloroform extractions. They obtained 30X coverage for reads longer than the “duplication” for one strain (UK 48) and had confusing results for the UK54 strain. They then identified a variable copy number of the same region in a population of UK54 cells. UK76 population contains cells with different levels of variation! Ring wants to study how quickly changes occur in mixed populations. Ring used ultra-long sequencing of a single colony and obtained the same level of variation. The group wants to investigate whether the rearrangements affect phenotype, including the motility of B. pertussis. This session was just what I needed today as I have been thinking about Delftia genomes. I wonder if some of the strains that have been difficult to sequence are highly variable?
