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High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions
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Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA

Zachary W Bent1, Mary B Tran-Gyamfi, Stanley A Langevin

  • 1Sandia National Laboratories, Livermore, CA 94551, USA.

Analytical Biochemistry
|March 29, 2013
PubMed
Summary

A new RNA capture technique significantly enriches pathogen transcripts from infected samples. This method improves pathogen gene analysis by increasing sequencing depth and coverage, crucial for understanding host-pathogen interactions.

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Area of Science:

  • Microbiology
  • Genomics
  • Infectious Diseases

Background:

  • Understanding host-pathogen interactions requires analyzing RNA transcripts from both organisms during infection.
  • Analyzing pathogen RNA is challenging due to the high abundance of host RNA in infected samples.
  • Second-generation sequencing often yields insufficient pathogen reads for effective gene-level analysis.

Purpose of the Study:

  • To develop and validate a novel capture-based technique for enriching pathogen transcripts.
  • To overcome the limitations of host RNA dominance in transcriptomic studies of infection.
  • To enable more comprehensive gene-level analysis of pathogens in infected hosts.

Main Methods:

  • A novel capture-based technique and device were developed to enrich pathogen transcripts.
  • Time-course tissue culture infections were performed using Rift Valley fever virus and Francisella tularensis.
  • RNA sequencing (RNA-Seq) was conducted on treated and untreated samples for comparative analysis.

Main Results:

  • The capture technique enriched pathogen reads by over an order of magnitude in all tested cases.
  • Significant increases were observed in the number of pathogen genes detected, their coverage, and sequencing depth.
  • The method demonstrated efficacy in enriching transcripts for both viral and bacterial pathogens.

Conclusions:

  • The novel capture technique substantially enhances the proportion of pathogen RNA in sequencing libraries.
  • This method significantly improves the ability to perform gene-level analysis of pathogens during infection.
  • The versatile technique holds promise for studying a wide range of host-pathogen systems.