Enrichment of Helminth Mitochondrial Genomes From Faecal Samples Using Hybridisation Capture.

Abstract

New approaches are urgently needed to enrich rare or low-abundant DNA in complex samples. Soil-transmitted helminths (STHs) inhabit heterogeneous environments, including the gastrointestinal tract of their host as adults and are excreted as eggs and larvae in faeces, complicating our understanding of their biology and the use of genetic tools for species monitoring and population tracking. We have developed a hybridisation capture approach to enrich mitochondrial genome sequences of two STH species, the roundworm Ascaris lumbricoides and whipworm Trichuris trichiura, from extracted DNA from faecal material and worm specimens. Employing ~1000 targeted probes, we achieved > 6000 and > 12,000 fold enrichment for A. lumbricoides and T. trichiura, respectively, relative to direct whole genome shotgun (WGS) sequencing. Sequencing coverage was highly concordant with probe targets and correlated with the number of eggs per gram (EPG) of parasites present, from which DNA from as few as 336 EPG for Ascaris and 48 EPG for Trichuris were efficiently captured and sufficient to provide effective mitochondrial genome data. Finally, allele frequencies were highly concordant between WGS and hybridisation capture, suggesting little genetic information is lost with additional sample processing required for enrichment. Our hybridisation capture design and approach enable sensitive and flexible STH mitochondrial genome sampling from faecal DNA extracts and pave the way for broader hybridisation capture-based genome-wide applications and molecular epidemiology studies of STHs.