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Lewis Stevens - Reference genomes from the field: the genome of Caenorhabditis bovis

Nanopore Community Meeting 2019

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Lewis Stevens opened his talk by stressing the significance of nematodes as parasites: ~1.5 billion people worldwide are currently infected with nematodes. He introduced Meloidogyne spp. which cause huge economic loss every year, and Onchocerca volvulvus, the organism responsible for river blindness. Whilst the nematode Caenorhabditis elegans has been extensively studied as a model organism, its applicability as a model for nematode parasitism is limited: in fact, all Caenorhabditis species currently in culture are free-living, and most were isolated from rotting fruits and flowers. In contrast, the species C. bovis is parasitic and “does things slightly differently”; it is responsible for parasitic otitis and in severe cases, mortality. Overall, Lewis described, we know very little about C. bovis – there’s just a handful of papers from the 1980s and 1990s that exist.

Lewis went on to explain how he contacted Eric Fevre, who had an initiative named ZooLink, a surveillance program at the International Livestock Research institute to study emerging zoonotic diseases in livestock. Thinking the process through, Lewis realised that it would be nearly impossible to take cattle samples out of the country of origin, and exporting extracted DNA would also have its challenges – so the only logical solution was attempt sequencing in situ. The plan, Lewis described, was to “go to Kenya, find the worm, sequence it” with a MinION.

When on site in Kenya, Lewis explained how the vets “did all the hard work of sticking fingers in cows’ ears”, but many samples contained nothing. Just when the team were giving up hope though, a worm crawled out of one of the samples!

In a field laboratory close to the sampling sites, C. bovis from the samples were cultured on horse blood agar. Lewis described the worms as “incredibly happy” on these plates – multiplying hugely in number and generating 8 μg of DNA from the extraction process. After extraction, the DNA was needle-sheared and then prepared for nanopore sequencing in a slightly modified "one pot" reaction using the Ligation Sequencing Kit. The read lengths of the two runs were very different, but this was attributed to the fact that for one sample, the DNA was “essentially cooked” in the rush to get it into solution.

The long reads were assembled via wtdbg2, corrected with Medaka and polished with the incorporation of short read data using Racon and Pilon.

The resulting C. bovis version 1 assembly spanned 62.7 Mb, with 35 contigs and a contig N50 of 7.6 Mb, reaching a BUSCO completeness of 95.2%. Half the genome was contained in 4 contigs, and two of those contigs represented complete chromosomes – “not bad for data generated in rural Kenya”. Phylogenetic analysis of the assembled genome revealed that C. bovis diverged early. Its closest relative in analysis was C. plicata, another ecologically unusual Caenorhabditis species: the nematode has been isolated from a dead elephant in Kenya and a dead pine marten in Germany, and spreads via carrion beetles. Given these results, Lewis asked: could there be a clade of vertebrate-associated Caenorhabditis species that is, as of yet, largely undiscovered?

Delving deeper into the C. bovis genome, Lewis noted that it exhibits low heterozygosity to the point of being essentially homozygous; he compared this to the C. benneri genome, which has been desribed as harbouring "the most molecular diversity of any eukaryote". Lewis suggested that the low diversity seen in the C. bovis genome results from the transport of a very small number of worms between parts of a population, resulting in extremely limited gene flow. He then discussed the presence of expansions in gene families associated with parasitism, with one potentially conferring resistance to antihelminthic drugs and others perhaps modulating the immune system of its mammalian hosts.

Finally, Lewis described his planned next steps for this study, highlighting how "to understand the biology of C. bovis, we need more than just a genome". To further study the worm, the team aim to export live cultures to the United Kingdom and to the Caenorhabditis Genetics Center (CGC) at the University of Minnesota. However, the biology of this worm in situ is still not fully understood, which means many more projects in Africa with local teams. Finally, Lewis thanked the teams supporting his project, in particular the vets who were “so willing to put their fingers in cows’ ears to look for a worm they’d never heard of”.

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