Authors

Discussion

Eimeria are a genus of Apicomplexan parasites which can infect all major livestock. Ingestion of this parasite leads to coccidiosis, an intestinal disease whose clinical signs include haemorrhagic diarrhoea, diminished weight gain, and mortality in severe infection. The genus is comprised of more than 1,500 species, each of which are monoxenous and mostly host-specific in their lifecycle. Of particular economic importance are species which infect chickens, as coccidiosis incurs costs upwards of £10.4 billion to annual global poultry production. Despite their impact in the agriculture sector, little is known about the genetic diversity of these parasites, and how this variation contributes to the rising level of resistance to current treatment strategies. Population genetic studies have typically utilised a select number of markers to infer evolutionary relationships between these parasites, and existing reference sequences remain incomplete due to both technical limitations and the inherent repetitive structure of Eimeria genomes.

This project combines the use of whole genome short-read sequencing and the most recent advancements in long-read sequencing technologies to overcome both technical and biological bottlenecks. We developed a novel Nanopore sequencing workflow to improve upon the quality of Eimeria reference genomes by generating long reads capable of spanning expansive regions of low complexity. Acting as scaffolds, these large sequences augment the de novo assembly of Eimeria genomes by properly orienting shorter sequences and bridging gaps between them. In doing so, we reduced the number of contigs in existing Eimeria genomes from 4,664 down to 250 whilst maintaining an equivalent level of completeness. Furthermore, these sequences were able to capture information within repeat-rich regions and sources of structural variation exceeding 1,000 base pairs in length, including insertions, deletions, inversions, repeat contractions, and repeat expansions.

Moreover, the availability of contiguous and complete reference sequences for Eimeria spp. was exploited in our whole genome short-read sequencing analysis of 18 Eimeria tenella clones selected for drug resistance and precocious development. Loci attributed to these phenotypes were identified for further investigation, including missense mutations, insertions, and deletions in genes coding for proteins involved in nucleotide binding, protein modification, and redox catalysis.

Our findings show that current Eimeria assemblies can be substantially improved upon with the use of Nanopore long reads. By resolving gaps and capturing low-complexity regions, more information is retained which benefits downstream analyses of whole genome sequencing data. Furthermore, the ability of long reads to retain large structural variants allows for more complete genotyping. The application of these methods to field samples may be used to enhance our understanding of the dynamics of these organisms, including the global distributions of different species, regional differences within and between species, as well as the genetic determinants of drug resistance.