Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples

Sci Rep. 2016 Oct 7;6:34850. doi: 10.1038/srep34850.

Abstract

Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont. While a suitable method is commercially available for mammalian samples of this type, development of such methods has languished for invertebrate samples. Furthermore, a common method across multiple taxa would facilitate comparisons between bacteria in invertebrate vectors and their vertebrate hosts. Here, a method is described to concurrently remove polyadenylated transcripts, prokaryotic rRNA, and eukaryotic rRNA, including those with low amounts of starting material (e.g. 100 ng). In a Wolbachia-Drosophila system, this bacterial mRNA enrichment yielded a 3-fold increase in Wolbachia mRNA abundance and a concomitant 3.3-fold increase in the percentage of transcripts detected. More specifically, 70% of the genome could be recovered by transcriptome sequencing compared to 21% in the total RNA. Sequencing of similar bacterial mRNA-enriched samples generated from Ehrlichia-infected canine cells covers 93% of the Ehrlichia genome, suggesting ubiquitous transcription across the entire Ehrlichia chaffeensis genome. This technique can potentially be used to enrich bacterial mRNA in many studies of host-microbe interactions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Brugia malayi / microbiology
  • Drosophila / microbiology
  • Ehrlichia chaffeensis / genetics
  • Genetic Techniques*
  • High-Throughput Nucleotide Sequencing / methods
  • Host-Pathogen Interactions / genetics*
  • Microfluidic Analytical Techniques
  • Poly A / chemistry
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / isolation & purification*
  • RNA, Messenger / chemistry
  • RNA, Messenger / isolation & purification*
  • RNA, Ribosomal / chemistry
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Symbiosis
  • Wolbachia / genetics
  • Wolbachia / pathogenicity
  • Wolves / microbiology

Substances

  • RNA, Bacterial
  • RNA, Messenger
  • RNA, Ribosomal
  • Poly A