Ribosomal RNA-depletion provides an efficient method for successful dual RNA-Seq expression profiling of a marine sponge holobiont

Abstract Investigations of host-symbiont interactions can benefit enormously from a complete and reliable holobiont gene expression profiling. The most efficient way to acquire holobiont transcriptomes is to perform RNA-Seq on both host and symbionts simultaneously. However, optimal methods for capturing both host and symbiont mRNAs are still under development, particularly when the host is a eukaryote and the symbionts are bacteria or archaea. Traditionally, poly(A)-enriched libraries have been used to capture eukaryotic mRNA, but the ability of this method to adequately capture bacterial mRNAs is unclear because of the short half-life of the bacterial transcripts. To determine the optimal RNA-Seq approach for animal host–bacterial symbiont analysis, we compared transcriptome bias, depth and coverage achieved by two different mRNA capture and sequencing strategies applied to the marine demosponge Amphimedon queenslandica holobiont, for which genomes of the animal host and three most abundant bacterial symbionts are available. The two strategies were: (i) poly(A) captured mRNA-Seq (Poly(A)-RNA-Seq); and (ii) ribosomal RNA depleted RNA-Seq (rRNA-depleted-RNA-Seq). For the host sponge, we find no significant difference in transcriptomes generated by the two different mRNA capture methods. However, for the symbiont transcriptomes, we confirm the expectation that the rRNA-depleted-RNA-Seq performs much better than the Poly(A)-RNA-Seq. Importantly, the availability of all genomes allowed us to accurately assess the nature of the Poly(A) bias compared to the rRNA-depleted transcriptomes. This comparison demonstrates that RNA-Seq by ribosomal RNA depletion is an effective and reliable method to analyse holobiont transcriptomes.