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

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.

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses

“…When targeting eukaryotic mRNA, polyadenylated RNA was isolated using the NEBNext Poly(A) mRNA magnetic isolation module. When targeting bacterial mRNA, samples underwent rRNA- and poly(A)-reductions, as previously described 10 , 14 . SPRIselect reagent (Beckman Coulter Genomics, Danvers, MA, USA) was used for cDNA purification between enzymatic reactions and size selection.…”

“…When extracting total RNA from a host sample, the signal from host transcripts typically overwhelms the signal from secondary organism transcripts under most biologically meaningful conditions. Differential enrichments have been designed to physically extract RNA from secondary organisms in samples by depleting highly abundant rRNAs and selecting transcripts based on differing properties between the two organisms, such as the differential poly-adenylation status of transcripts in the case of samples containing a mixture of eukaryotic and prokaryotic RNA 10 . However, in eukaryote-eukaryote dual species transcriptomics experiments, these differences are usually not present to be exploited.…”

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses

“…When targeting eukaryotic mRNA, polyadenylated RNA was isolated using the NEBNext Poly(A) mRNA magnetic isolation module. When targeting bacterial mRNA, samples underwent rRNA- and poly(A)-reductions, as previously described [10, 14]. SPRIselect reagent (Beckman Coulter Genomics, Danvers, MA, USA) was used to purify cDNA between enzymatic reactions and perform size selection.…”

“…When extracting total RNA from a host sample, the signal from host transcripts typically overwhelms the signal from secondary organism transcripts under most biologically meaningful conditions. Differential enrichments have been designed to physically extract RNA from secondary organisms in samples by depleting the highly abundant rRNAs and selecting transcripts based on differing properties between the two organisms, such as the differential poly-adenylation status of transcripts [10]. However, in eukaryote-eukaryote dual species transcriptomics experiments, these differences are often not present.…”

Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics

“…Since mRNAs can comprise just 1-20% of the total RNA extract [12,95,96], enrichment of mRNAs, for instance by rRNA depletion, is often carried out prior to RT in order to improve coverage of protein coding regions (methods for enrichment are reviewed in [5] and evaluated in [97]). Enrichment may also be necessary when using host-bacterial mixed samples in which the host RNAs will also contaminate the sample [85,98]. However, this enrichment step is not always necessary; for instance, dRNA-Seq (described in Section 5.1) is often performed without enrichment in order to study the whole transcriptome [99].…”