Enriching Bacteria-Specific RNA From Host Samples Before NGS With Transcript-Capture

Pathogen gene expression from host samples is often challenging to study due to low signal and high host RNA background. PCR probes have been recently used to hybridize and extract bacterial sequences from next-generation sequencing (NGS) libraries generated from in vitro and animal models of infection; however, these strategies require purchasing commercially synthesized probes that often do not capture the entire transcriptome. Transcript-capture sequencing is a novel capture approach for extracting RNA of a target bacterial species from samples in which there is substantial contamination by the host or other microbes. Biotinylated 150-base-pair DNA probes are generated in-house from bacterial DNA spanning the entire bacterial genome. Probes are hybridized to the cDNA of NGS sequencing libraries prepared from host samples to capture and enrich for bacterial-specific RNA reads before sequencing. This method results in a >200-fold increase in bacterial RNA reads from infected host samples (including in vitro, animal, and human samples) and generates complete bacterial transcriptomes with high gene coverage (>80%). Use of this protocol on infected host samples reveals a snapshot of bacterial activity during disease that may improve understanding of the physiological state of pathogens within their hosts. Key features • Generates single-stranded biotinylated DNA probes in-house from bacterial gDNA. • Uses DNA probes in a hybridization capture approach to enrich for cDNA of a target species >200-fold in standard next-generation RNA sequencing libraries. • Allows generation of complete bacterial transcriptomes (>80% gene coverage) from samples in which transcriptomic signal-to-noise is limiting, such as from animal models and clinical samples.