{ "abstract": "Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity (low and standard laboratory doses) and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infection were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author summary: Soil-transmitted helminths (STHs), such as the parasite Strongyloides, are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STHs in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in faecal samples from humans infected with STHs, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read (SR) Illumina and long-read (LR) Oxford Nanopore Technologies, but SR outperformed LR shotgun metagenomics. Contamination of bacterial sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.", "abstract_shown": true, "affiliations": [ "Life Sciences Department, University of Bath, Bath, United Kingdom", "Forestry Vocational School, Düzce University, Konuralp Campus, Düzce, Türkiye", "Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom", "Academic Department of Military Medicine, Royal Centre for Defence Medicine, Birmingham, United Kingdom", "Department of Evolution, Ecology and Behaviour, University of Liverpool, Liverpool, United Kingdom", "Tropical Disease Research Center, Department of Tropical Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand", "Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Australia" ], "article_type": "Research Article", "arxiv": null, "author_total": 15, "authors": [ { "affs": [ 0 ], "name": "Katie O’Brien" }, { "affs": [ 1 ], "name": "Ajay Elamaran" }, { "affs": [ 2 ], "name": "Mehmet Dayi" }, { "affs": [ 3 ], "name": "Georgia Keeling" }, { "affs": [ 4 ], "name": "William D. Nevin" }, { "affs": [ 5 ], "name": "Yuchen Liu" }, { "affs": [ 6 ], "name": "Mark Viney" }, { "name": "Kieran Reynolds" }, { "name": "Charmaine Bishop" }, { "name": "Banchob Sripa" }, { "name": "Menebere Woubshete" }, { "name": "Pamela Sachs Nique" }, { "name": "Rhiannon Wright" }, { "name": "Jane Younger" }, { "name": "Vicky L. Hunt" } ], "bibtex": "https://research.draconitic.com/paper/7444f3v41ng5x.bib", "cited_by": 0, "doi": "10.1371/journal.pntd.0014130", "fulltext_url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014130", "id": "7444f3v41ng5x", "issns": [ "1935-2735" ], "landing_url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014130", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2405|Parasites and Host Interactions" ], [ "plos", "/Biology and life sciences/Biochemistry/Bioenergetics/Energy-producing organelles/Mitochondria" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular structures and organelles/Energy-producing organelles/Mitochondria" ], [ "plos", "/Biology and life sciences/Computational biology/Genome analysis/Genomic databases" ], [ "plos", "/Biology and life sciences/Genetics/Genomics" ], [ "plos", "/Biology and life sciences/Genetics/Genomics/Genome analysis/Genomic databases" ], [ "plos", "/Biology and life sciences/Genetics/Genomics/Metagenomics" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Cloning/DNA cloning/Shotgun sequencing" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Sequencing techniques/Shotgun sequencing" ], [ "plos", "/Medicine and health sciences/Medical conditions/Parasitic diseases" ], [ "plos", "/Medicine and health sciences/Medical conditions/Parasitic diseases/Helminth infections/Soil-transmitted helminthiases" ], [ "plos", "/Medicine and health sciences/Medical conditions/Parasitic diseases/Nematode infections" ], [ "plos", "/Medicine and health sciences/Medical conditions/Tropical diseases/Neglected tropical diseases/Soil-transmitted helminthiases" ], [ "plos", "/Research and analysis methods/Database and informatics methods/Biological databases/Genomic databases" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Cloning/DNA cloning/Shotgun sequencing" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Sequencing techniques/Shotgun sequencing" ] ], "openalex": "W7213881260", "pdf_url": "https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0014130&type=printable", "pmcid": null, "pmid": null, "published": "2026-09-21", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/7444f3v41ng5x.ris", "sources": [ { "first_seen": "2026-10-30T12:17:43Z", "native_id": "10.1371/journal.pntd.0014130", "source": "plos", "url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014130" } ], "subjects": [ { "field": "biochemistry", "origin": "plos:molecular biology", "subfield": "molecular-biology" }, { "field": "biology", "origin": "plos:genomics", "subfield": "genetics-genomics" } ], "title": "Hunting for Helminths: short- and long-read shotgun metagenomics for helminth detection in faecal samples", "title_text": "Hunting for Helminths: short- and long-read shotgun metagenomics for helminth detection in faecal samples", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:43Z", "url": "https://research.draconitic.com/paper/7444f3v41ng5x", "venue": "PLOS Neglected Tropical Diseases", "versions": [] }