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Here, we develop Transcript-Capture Seq, which uses customizable biotinylated probes generated in-house to enrich bacteria-specific RNA from host samples before Next Generation Sequencing (NGS). This method results in a &amp;gt;200-fold increase in bacterial mRNA reads from mixed samples and allows analysis of the complete bacterial transcriptome from clinical samples. We apply Transcript-Capture to models of tuberculosis (TB) infection as well as sputum samples from TB patients. TB patients exhibit unexplained heterogeneity in disease progression, and the activity of Mycobacterium tuberculosis (Mtb) has been proposed to affect treatment response. By applying Transcript-Capture to sputum samples collected from TB patients, we generate the first complete in vivo bacterial transcriptome of Mtb via NGS. Mtb from patient sputa shows upregulation of genes involved in host lipid utilization and zinc limitation, as well as a similar gene expression profile to Mtb log phase growth in vitro. Applying Transcript-Capture to clinical sputa provides a snapshot of bacterial activity directly from human patients and can be used to investigate the physiological state of bacteria surviving in vivo.<\/jats:p>","DOI":"10.1093\/narmme\/ugaf045","type":"journal-article","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T12:56:18Z","timestamp":1766580978000},"source":"Crossref","is-referenced-by-count":2,"title":["Transcript-Capture sequencing enriches mRNA of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    from host samples"],"prefix":"10.1093","volume":"3","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6537-7755","authenticated-orcid":false,"given":"Eleanor I","family":"Lamont","sequence":"first","affiliation":[{"name":"Department of Microbiology, University of Washington , Seattle, WA 98195 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard 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Molecular Medicine, University of Cape Town , Observatory 7925 ,","place":["Republic of South Africa"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Yu","sequence":"additional","affiliation":[{"name":"Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health , Bethesda, MD 20892 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle M","family":"Weiner","sequence":"additional","affiliation":[{"name":"Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health , Bethesda, MD 20892 ,","place":["United States"]},{"name":"Tuberculosis Imaging Program, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health , Bethesda, MD 20892 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura E","family":"Via","sequence":"additional","affiliation":[{"name":"Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health , Bethesda, MD 20892 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jill","family":"Winter","sequence":"additional","affiliation":[{"name":"Catalysis Foundation for Health , San Ramon, CA 94608 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0783-1328","authenticated-orcid":false,"given":"Shuyi","family":"Ma","sequence":"additional","affiliation":[{"name":"Center for Global Infectious Disease Research, Seattle Children\u2019s Research Institute , Seattle, WA 98101 ,","place":["United States"]},{"name":"Department of Pediatrics, University of Washington , Seattle, WA 98195 ,","place":["United States"]},{"name":"Department of Chemical Engineering, University of Washington , Seattle, WA 98195 ,","place":["United States"]},{"name":"Department of Global Health, University of Washington , Seattle, WA 98195 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert\u00a0J","family":"Wilkinson","sequence":"additional","affiliation":[{"name":"Institute of Infectious Disease and Molecular Medicine, University of Cape Town , Observatory 7925 ,","place":["Republic of South Africa"]},{"name":"Francis Crick Institute Midland Road , London, NW1 1AT ,","place":["UK"]},{"name":"Department of Infectious Diseases, Imperial College London W12 0NN ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"Clifton 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