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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 &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\n                  <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                  (Mtb) has been proposed to affect treatment response. By applying Transcript-Capture to sputum samples collected from TB patients we generate the first complete\n                  <jats:italic>in vivo<\/jats:italic>\n                  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\n                  <jats:italic>in vitro<\/jats:italic>\n                  . 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\n                  <jats:italic>in vivo<\/jats:italic>\n                  .\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>GRAPHICAL ABSTRACT<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"685133v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.10.28.685133","type":"posted-content","created":{"date-parts":[[2025,10,29]],"date-time":"2025-10-29T03:35:19Z","timestamp":1761708919000},"source":"Crossref","is-referenced-by-count":2,"title":["Transcript-Capture sequencing enriches mRNA of\n                  <i>Mycobacterium tuberculosis<\/i>\n                  from host samples"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6537-7755","authenticated-orcid":false,"given":"Eleanor I.","family":"Lamont","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard M.","family":"Jones","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Assadi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"Morrison","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taeksun","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle M.","family":"Weiner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura E.","family":"Via","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jill","family":"Winter","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3347-3491","authenticated-orcid":false,"given":"Shuyi","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert J.","family":"Wilkinson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"Clifton E.","family":"Barry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David R.","family":"Sherman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025103021001223000_2025.10.28.685133v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2852"},{"key":"2025103021001223000_2025.10.28.685133v1.2","doi-asserted-by":"publisher","DOI":"10.1021\/ACSINFECDIS.8B00369"},{"key":"2025103021001223000_2025.10.28.685133v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-022-00731-y"},{"key":"2025103021001223000_2025.10.28.685133v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.12.033"},{"key":"2025103021001223000_2025.10.28.685133v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-40055-1"},{"key":"2025103021001223000_2025.10.28.685133v1.6","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02363-23"},{"key":"2025103021001223000_2025.10.28.685133v1.7","doi-asserted-by":"publisher","DOI":"10.15252\/MSB.20188584"},{"key":"2025103021001223000_2025.10.28.685133v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-23276-8"},{"key":"2025103021001223000_2025.10.28.685133v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-020-0683-3"},{"key":"2025103021001223000_2025.10.28.685133v1.10","doi-asserted-by":"publisher","DOI":"10.1126\/SCITRANSLMED.ADK8222"},{"key":"2025103021001223000_2025.10.28.685133v1.11","doi-asserted-by":"publisher","DOI":"10.1128\/MBIO.01424-22"},{"key":"2025103021001223000_2025.10.28.685133v1.12","doi-asserted-by":"publisher","DOI":"10.12688\/gatesopenres.12750.1"},{"key":"2025103021001223000_2025.10.28.685133v1.13","doi-asserted-by":"publisher","DOI":"10.1007\/s15010-009-8450-7"},{"key":"2025103021001223000_2025.10.28.685133v1.14","unstructured":"World Health Organization. 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