Schistosoma Species and Hybrid Genotyping With a Field Deployable Multi-Marker Amplicon Panel

Schistosomes are parasitic trematode worms of the genus Schistosoma, responsible for causing urogenital and intestinal schistosomiasis. Six primary species infect humans: S. mansoni, S. japonicum, S. mekongi, S. intercalatum, S. guineensis, and S. haematobium. In addition, several species including S. bovis, S. curassoni, and S. mattheei primarily infect animals, particularly cattle. Hybridization events have been documented both between human-infecting species and between human- and animal-infecting species of Schistosoma. Current methods for detecting hybrids rely on genotyping mitochondrial cytochrome oxidase I (cox1) and the ribosomal internal transcribed spacer (ITS), or whole-genome sequencing. This protocol describes a multi-locus, multiplex amplicon sequencing approach using Nanopore technology, referred to as NMAS-Seq, for species-level genotyping and hybrid detection. It includes comprehensive steps for multiplex nested PCR, amplicon quantification and pooling, library preparation, and sequencing. The workflow is adaptable for molecular typing of other parasitic species. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA extraction and multiplex nested PCR Basic Protocol 2: Amplicon quantification and pooling Basic Protocol 3: Amplicon library preparation and Nanopore sequencing.