Developing a Bioinformatic Workflow to Analyze Metagenomes of the Vibrio-Squid Symbiosis

Host-colonization sites containing low-diversity microbial populations present a unique problem when applying metagenomic approaches. For example, how does one extract information such as the number of unique genotypes and relative abundance of symbiotically important genes? The light organ of the Hawaiian bobtail squid (Euprymna scolopes) is colonized by multiple strains of a bioluminescent bacterial species, Vibrio fischeri, making the light organ a naturally occurring low-diversity system. Studying the strain-level diversity of V. fischeri is of great interest as it reveals conserved and unique strategies to compete and colonize a host. Thus, applying metagenomics to this symbiosis model system is critical to further our understanding of such genetic factors and the eco-evolutionary dynamics in symbiotic associations. To achieve this goal, we set out to develop a method for assessing strain-level genomic variability of the V. fischeri light organ community. By using synthetic “mock” communities with mixed proportions of six sequenced V. fischeri strains, we first tested the limit of detection of sequencing methods. To analyze the metagenome sequences, we created a bioinformatics pipeline optimized for use with closely related strains. This analysis allows us to make predictions about community composition, number of ancestral genomes, and the relative abundance of genes of interest, including the type VI secretion system II (T6SS2), an interbacterial weapon known to be strain-specific in adult light organs. After validating our approach with our mock communities, we applied our approach to samples from the light organ community of a wild-caught adult E. scolopes. Ultimately, we created an adaptable bioinformatics workflow capable of filling gaps in our knowledge about V. fischeri strain-level diversity during symbiotic association.