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

Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; Hamblin, Rachelle; Williams, Kelly P.; Meagher, Robert J.; Chang, ed., Yung-Fu

doi:10.1371/journal.pone.0168788

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

Journal Article · Wed Dec 21 00:00:00 EST 2016 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0168788· OSTI ID:1338433

Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; Hamblin, Rachelle; Williams, Kelly P.;

; Chang, ed., Yung-Fu

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.

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Research Organization:: Sandia National Lab. (SNL-CA), Livermore, CA (United States)

Sponsoring Organization:: USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: 173021; AC04-94AL85000

OSTI ID:: 1338433

Alternate ID(s):: OSTI ID: 1303158

Report Number(s):: SAND2016-4584J; 10.1371/journal.pone.0168788

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Vol. 11 Journal Issue: 12; ISSN 1932-6203

Publisher:: Public Library of Science (PLoS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 4 works

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

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