Transcript-Capture sequencing enriches mRNA of <i>Mycobacterium tuberculosis</i> from host samples

Bacterial gene expression from sites of infection are poorly studied due to low levels of bacterial mRNA present in clinical samples. 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 >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 <i>Mycobacterium tuberculosis</i> (Mtb) has been proposed to affect treatment response. By applying Transcript-Capture to sputum samples collected from TB patients, we generate the first complete <i>in vivo</i> 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 <i>in vitro</i>. 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 <i>in vivo</i>.