(125a) Efficient Bacterial mRNA Sequencing in Diverse Species and Co-Cultures

Microbes in nature remain one of the largest untapped resources for novel bio-based products, including fuels, antibiotics, and waste treatment solutions. Delineating the transcriptomes of these species with RNA-seq would provide powerful datasets that could be used to characterize the cellular and biochemical functions within these species. However, a major challenge in bacterial RNA-seq is that highly abundant ribosomal RNA (rRNA) molecules must be removed prior to sequencing. If left undepleted, rRNA can consume up to 90% of the sequencing data with uninformative reads, resulting in highly inefficient sequencing of messenger RNA (mRNA), that encodes proteins. This work presents a method, EMBR-seq, which depletes 5S, 16S, and 23S rRNA in prokaryotic species through a combination of selective polyadenylation on mRNA, blocking of rRNA-derived cDNA amplification, and targeted RNase H activity on rRNA. Compared to alternate rRNA depletion methods that employ a large array of oligonucleotides, EMBR-seq employs a few short oligonucleotides per rRNA subunit at strategic locations, thereby making this new technology significantly cheaper and simpler to optimize for any specific prokaryotic species. We demonstrate that rRNA content is depleted down to 3% of sequenced reads in E. coli, 1% in G. metallireducens, and 9% in Fibrobacter sp. UWB7. Additionally, we apply EMBR-seq in co-cultures of Fibrobacter sp. UWB7 with anaerobic fungi, to efficiently obtain RNA-seq data from mixed prokaryotic-eukaryotic samples and quantify the transcriptional changes compared to monoculture growth. This analysis deepens our understanding of the relationship between two species native to the rumen microbiome and how co-culture conditions induce changes in the bacterial stress response and their cellulolytic activity. Thus, EMBR-seq provides an efficient approach to perform RNA-seq quantification of diverse bacterial systems.