A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing.

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000-3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.
Competing Interests: The authors have declared that no competing interests exist. The authors are current or former employees of Sandia National Laboratories. The specific roles of these authors are articulated in the ‘author contributions’ section. Sandia National Laboratories did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing interests related to their employment with Sandia National Laboratories. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Author ZWB is presently an employee of 10X Genomics. The authors attest that 10X Genomics had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing interests related to ZWB’s present employment with 10X Genomics.