Development and validation of a multilocus sequence typing scheme for Fasciola hepatica using next-generation deep amplicon sequencing

Fasciolosis, caused by Fasciola hepatica, is an economically important disease in sheep and cattle. Despite its importance, the population structures of F. hepatica are less known at the nuclear DNA level. Knowledge of the population genetic structure of F. hepatica is important for informing disease control. We designed, developed, and validated a multilocus sequence typing (MLST) scheme based on six markers. These markers were selected by aligning newly sequenced whole-genome sequence (WGS) data with available reference genomes and selecting variable regions with five or more SNPs from different scaffolds of the F. hepatica reference genome Fasciola 10x pilon (GCA_900302435.1). Initially, 20 markers were identified, of which 18 were validated. Of which 12 were multiplexed for deep amplicon sequencing after validation on worm and faecal eggs DNA; however, six markers were finally retained for downstream population genetics analysis. These markers were used to investigate genetic diversity and host-associated genetic structuring in cattle- and sheep-derived F. hepatica populations in UK samples. A total of 53 unique alleles from six MLST markers were identified from 30 faecal (cattle = 13, sheep = 17) and 12 adult worm (cattle = 2, sheep = 10) populations. Shared alleles were observed in sheep- and cattle-derived populations. The highest allele diversity was observed in the Scottish Borders, Southern Scotland, and South-West England, and the lowest in North-West England. Minimal genetic differentiation was observed between cattle- and sheep-derived populations, with most genetic structuring within rather than between populations. Five markers showed high polymorphism, whereas one marker showed low polymorphism, highlighting the importance of multilocus approaches. Overall, this six MLST-marker panel provides a tool for population genetic studies, revealing high gene flow and clonal population structure of F. hepatica across hosts and regions in the UK.Keywords: Fasciola hepatica, MLST, population genetics, SNP, polymorphism, Deep amplicon sequencing