Abstract

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.

Highlights

  • Many filarial nematode species responsible for debilitating human diseases contain Wolbachia, an obligate intracellular endosymbiotic bacterium

  • Prior sequencing of total RNA from different lifecycle stages of a similar filarial nematode, Dirofilaria immitis, yielded ~0.7% average reads mappable to Wolbachia (0.02–2.11% depending on life-cycle stage) [10]

  • In the present study between 1.3% and 1.6% (MF) of reads from B. malayi total RNA mapped to the Wolbachia from B. malayi (wBm) genome (Table 1)

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Summary

Introduction

Many filarial nematode species (including Wuchereria bancrofti, Brugia malayi, B. timori and Onchocerca volvulus) responsible for debilitating human diseases contain Wolbachia, an obligate intracellular endosymbiotic bacterium. Wolbachia are found in the lateral cords of all adult worms as well as within the oocytes and developing embryos in the female reproductive tract [1,2,3,4]. Treatment with tetracycline antibiotics depletes Wolbachia leading to decreased fertility and eventually death of adult worms [5,6,7]. Data collection and analysis, decision to publish, or preparation of the manuscript. Critical feedback and proofreading of the manuscript was provided by additional members of the NEB

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