{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:28:43Z","timestamp":1787059723708,"version":"build-2736575974"},"reference-count":35,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T00:00:00Z","timestamp":1787011200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T00:00:00Z","timestamp":1787011200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001592","name":"Higher Education Authority","doi-asserted-by":"publisher","award":["AICRI-Start"],"award-info":[{"award-number":["AICRI-Start"]}],"id":[{"id":"10.13039\/501100001592","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100012921","name":"Royal College of Surgeons in Ireland","doi-asserted-by":"publisher","award":["StAR International PhD scholarship"],"award-info":[{"award-number":["StAR International PhD scholarship"]}],"id":[{"id":"10.13039\/100012921","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100012921","name":"Royal College of Surgeons in Ireland","doi-asserted-by":"publisher","award":["StAR MD scholarship"],"award-info":[{"award-number":["StAR MD scholarship"]}],"id":[{"id":"10.13039\/100012921","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSystems"],"published-print":{"date-parts":[[2026,8,18]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      The study of tumor-associated microbiomes using whole-genome sequencing (WGS) has attracted considerable attention, but microbial signal detection remains controversial due to host contamination and methodological artifacts. As the necessity of human-read removal becomes increasingly evident, many groups now include this step in their data pre-processing workflows. In this work, we introduce an open-source tool, HumanFilt, designed for rigorous host-read removal and apply it to the re-analysis of WGS data from 10 mucinous rectal adenocarcinoma cases originally published by Reynolds et al. The workflow integrates k-mer-based classification (Kraken2), quality and adapter trimming (Trim Galore), vector filtering (BBDuk\/UniVec_Core), and duplicate removal (FastUniq). After reducing data complexity, a multi-aligner, multi-reference approach (BWA-MEM\/GRCh38, Bowtie2\/T2T-CHM13, and Minimap2\/Human Pangenome Reference Consortium v1.1) removes remaining host sequences, collectively eliminating more than 99.9% of human-derived reads. Although the additional alignment steps eliminated only a small fraction of total reads, they consistently removed millions of residual sequences per sample, underscoring the importance of rigorous filtering in data sets where non-human reads are a small minority. Taxonomic profiling with PathSeq and MetaPhlAn revealed reproducible enrichment of\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      species in tumor versus matched normal tissues, and comparison before and after filtering showed that this tumor-over-normal pattern was preserved despite an overall reduction in RPM values. Simulation analyses further showed that HumanFilt preserved more than 99.7% of true\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      signals, supporting high specificity without meaningful false-negative loss of microbial reads. In direct comparison with Deacon and NoHuman, HumanFilt achieved the most stringent host-read removal but also removed a greater proportion of PathSeq-classified\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      reads, highlighting the trade-off between maximal host depletion and preservation of ambiguous microbial signal. Cross-validation with immunofluorescence analysis using pan-\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      (detecting both\n                      <jats:italic toggle=\"yes\">Fusobacterium animalis<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Fusobacterium nucleatum<\/jats:italic>\n                      ) and\n                      <jats:italic toggle=\"yes\">F. nucleatum<\/jats:italic>\n                      -specific antibodies showed general consistency with\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      subspecies detected by WGS. Compared to the unfiltered analysis, host depletion markedly reduced artificial microbial signals in normal samples while preserving tumor-associated\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      , resulting in a more reliable microbial profile.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      We developed an open-source tool that enables rapid removal of human-derived sequences and applied it to rectal cancer whole-genome sequencing data. This approach reduced false microbial signals while preserving true tumor-associated\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      , and simulation analyses showed that it retained more than 99.7% of true\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      reads. Comparison with Deacon and NoHuman showed that HumanFilt achieved more stringent host depletion but also highlighted the trade-off between aggressive host filtering and preservation of ambiguous microbial signal. We also observed general consistency between the sequencing results and immunofluorescence staining in tissue. Together, these findings provide a more reliable basis for studying tumor-bacteria interactions.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/msystems.00550-26","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T13:03:16Z","timestamp":1784034196000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["HumanFilt: a multi-reference host depletion pipeline improves\n                    <i>Fusobacterium<\/i>\n                    detection accuracy in tumor WGS data sets"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0236-8573","authenticated-orcid":false,"given":"Mariia","family":"Frolova","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01hxy9878","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Physiology and Medical Physics and RCSI Centre for Systems Medicine, Royal College of Surgeons in Ireland","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barry","family":"Maguire","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01hxy9878","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Physiology and Medical Physics and RCSI Centre for Systems Medicine, Royal College of Surgeons in Ireland","place":["Dublin, Ireland"]},{"id":[{"id":"https:\/\/ror.org\/043mzjj67","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Colorectal Surgery, Beaumont Hospital","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heiko","family":"Duessmann","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01hxy9878","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Physiology and Medical Physics and RCSI Centre for Systems Medicine, Royal College of Surgeons in Ireland","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Menatallah","family":"Rayan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01hxy9878","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Physiology and Medical Physics and RCSI Centre for Systems Medicine, Royal College of Surgeons in Ireland","place":["Dublin, Ireland"]},{"id":[{"id":"https:\/\/ror.org\/00yhnba62","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pharmaceutical Sciences, College of Pharmacy, QU Health, Qatar University","place":["Doha, Qatar"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caoimbhe","family":"Burke","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05m7pjf47","id-type":"ROR","asserted-by":"publisher"}],"name":"UCD School of Medicine, UCD Conway Institute, University College Dublin","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hesham M.","family":"Korashy","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00yhnba62","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pharmaceutical Sciences, College of Pharmacy, QU Health, Qatar University","place":["Doha, Qatar"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arman","family":"Raman","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05m7pjf47","id-type":"ROR","asserted-by":"publisher"}],"name":"UCD School of Medicine, UCD Conway Institute, University College Dublin","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William M.","family":"Gallagher","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05m7pjf47","id-type":"ROR","asserted-by":"publisher"}],"name":"UCD School of Medicine, UCD Conway Institute, University College Dublin","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John P.","family":"Burke","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043mzjj67","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Colorectal Surgery, Beaumont Hospital","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon J.","family":"Furney","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01hxy9878","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Physiology and Medical Physics and RCSI Centre for Systems Medicine, Royal College of Surgeons in Ireland","place":["Dublin, Ireland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3479-7794","authenticated-orcid":true,"given":"Jochen H. 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Trim Galore. Available from: https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/trim_galore\/. Retrieved 24 October 2025."},{"key":"e_1_3_5_35_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41388-024-02974-w"},{"key":"e_1_3_5_36_2","doi-asserted-by":"publisher","DOI":"10.1158\/1940-6207.CAPR-16-0178"}],"container-title":["mSystems"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msystems.00550-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msystems.00550-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:02:50Z","timestamp":1787058170000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/msystems.00550-26"}},"subtitle":[],"editor":[{"given":"Germ\u00e1n","family":"Bonilla-Rosso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,18]]},"references-count":35,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8,18]]}},"alternative-id":["10.1128\/msystems.00550-26"],"URL":"https:\/\/doi.org\/10.1128\/msystems.00550-26","relation":{},"ISSN":["2379-5077"],"issn-type":[{"value":"2379-5077","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,18]]},"assertion":[{"value":"2026-04-29","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-06-18","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-14","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00550-26"}}