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However, microbial contamination and host-derived DNA remain a major obstacle to accurate data interpretation. Here, we present a methodology called \u2018Stop-Check-Go\u2019 for detecting and mitigating contaminants in metagenomic datasets obtained from neonatal patient samples (nasal and rectal swabs). This method incorporates laboratory and bioinformatics work combining a prevalence method, coverage estimation and microbiological reports. We compared the \u2018Stop-Check-Go\u2019 decontamination system with other published decontamination tools and commonly found poor performance in decontaminating microbiologically negative patients (false positives). We emphasize that host DNA decreased by an average of 76% per sample using a lysis method and was further reduced during post-sequencing analysis. Microbial species were classified as putative contaminants and assigned to \u2018Stop\u2019 in nearly 60% of the dataset. The \u2018Stop-Check-Go\u2019 system was developed to address the specific need of decontaminating low-biomass samples, where existing tools primarily designed for short-read metagenomic data showed limited performance.<\/jats:p>","DOI":"10.1099\/mgen.0.001609","type":"journal-article","created":{"date-parts":[[2026,1,22]],"date-time":"2026-01-22T15:17:04Z","timestamp":1769095024000},"source":"Crossref","is-referenced-by-count":0,"title":["Mitigation and detection of putative microbial contaminant reads from long-read metagenomic datasets"],"prefix":"10.1099","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-1407-4397","authenticated-orcid":false,"given":"Stefany","family":"Ayala-Monta\u00f1o","sequence":"first","affiliation":[{"name":"Institute for Infection Prevention and Control, Medical Centre \u2013 University of Freiburg, Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ayorinde O.","family":"Afolayan","sequence":"additional","affiliation":[{"name":"Institute for Infection Prevention and Control, Medical Centre \u2013 University of Freiburg, Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raisa","family":"Kociurzynski","sequence":"additional","affiliation":[{"name":"Institute for Infection Prevention and Control, Medical Centre \u2013 University of Freiburg, Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ulrike","family":"Loeber","sequence":"additional","affiliation":[{"name":"Charit\u00e9 \u2013 Universit\u00e4tsmedizin Berlin, Berlin, Germany"},{"name":"Experimental and Clinical Research Center (ECRC), a cooperation between the Max\u2010Delbr\u00fcck\u2010Center for Molecular Medicine in the Helmholtz Association and the Charit\u00e9, Berlin, Germany"},{"name":"Max Delbr\u00fcck Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany"},{"name":"German Centre for Cardiovascular Research (DZHK), Partner Site Berlin, Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1672-5789","authenticated-orcid":false,"given":"Sandra","family":"Reuter","sequence":"additional","affiliation":[{"name":"Institute for Infection Prevention and Control, Medical Centre \u2013 University of Freiburg, Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"345","reference":[{"key":"R1","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2021.641921"},{"key":"R2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-022-02619-9"},{"key":"R3","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-014-0087-z"},{"key":"R4","doi-asserted-by":"publisher","DOI":"10.1128\/mSystems.00186-19"},{"key":"R5","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1006967"},{"key":"R6","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-025-02035-2"},{"key":"R7","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-018-0605-2"},{"key":"R8","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-023-01477-2"},{"key":"R9","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2018.11.003"},{"key":"R10","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1650"},{"key":"R11","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-023-01696-w"},{"key":"R12","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-023-01737-5"},{"key":"R13","doi-asserted-by":"publisher","DOI":"10.1186\/s12967-024-04917-1"},{"key":"R14","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2021.600111"},{"key":"R15","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-021-01012-1"},{"key":"R16","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.766364"},{"key":"R17","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.745076"},{"key":"R18","doi-asserted-by":"publisher","DOI":"10.3390\/mi8030083"},{"key":"R19","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-00746-x"},{"key":"R20","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty191"},{"key":"R21","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp352"},{"key":"R22","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3820"},{"key":"R23","article-title":"Workflow to remove adapter from uncorrected long reads, filter reads, assemble genomes, and error-correct the assemblies. 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