Developing long-read Oxford Nanopore nemabiome metabarcoding for ovine gastrointestinal nematode community analysis and diagnostics

Parasitic gastrointestinal nematodes (GIN) are major causes of disease in animals and humans and the routine use of anthelmintic drugs in their management is increasingly ineffective due to widespread anthelmintic drug resistance. Consequently, there is a growing need for improved diagnostic tools to enable more evidence-based approaches to control including more targeted and sustainable drug use. ITS-2 nemabiome metabarcoding is a powerful method to determine the relative abundance of GIN species isolated from fecal samples from a number of animal species. This thesis presents research aiming to improve this method to increase its flexibility, widen its applicability and make it more suitable for more routine diagnostics and surveillance. Sheep are an excellent system to develop GIN molecular diagnostic methods; GIN are arguably better studied in sheep than in any other host due to their major impact on sheep production. The research in this thesis has three main aims: (i) To transition nemabiome metabarcoding away from the short-read Illumina platform to the long-read Oxford Nanopore Technologies (ONT) platform. (ii) To apply nemabiome metabarcoding directly to DNA from fecal material rather than harvested parasite populations. (iii) To develop nemabiome metabarcoding to determine the absolute abundance of GIN in fecal samples rather than just their relative abundance. Chapter 2 presents a set of four new non-redundant nematode rRNA databases and a WebApp, ‘PrimerTC’, to assess the taxonomic conservation of primers targeting different regions of the rRNA cistron across the nematode phylum. These new resources should enable more targeted and more comprehensive metabarcoding for both parasitic and free-living nematodes. Chapter 3 introduces ITS-1/5.8S/ITS-2 rRNA long-read ONT metabarcoding for GIN species relative quantitation. This chapter also pioneers the application of ITS-2 and ITS-1/5.8S/ITS-2 metabarcoding directly to DNA extracted from ovine fecal samples. Chapter 4 explores a synthetic DNA spike-in approach for absolute quantitation of GIN species in ovine fecal samples using both ITS-2 rDNA Illumina short-read and ITS-1/5.8S/ITS-2 long-read nemabiome metabarcoding. The new tools and methodologies presented in this thesis should pave the way for more widescale adoption of nemabiome metabarcoding as a tool in research, surveillance, and routine diagnostics.
Next Generation Sequencing, Nemabiome Metabarcoding, Gastrointestinal Nematodes, Nematode Databases, Long read sequencing, Nematodes Diagnostics
Charrier, E. (2024). Developing long-read Oxford Nanopore nemabiome metabarcoding for ovine gastrointestinal nematode community analysis and diagnostics (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from