Author: Qi Yang
Yang, Qi, 2017 Identifying untapped microbial resources in the marine sponge microbiome, Flinders University, School of Medicine
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Marine sponge (phylum Porifera) associated microorganisms are functionally and economically valuable for marine benthic ecosystem and natural bioactive compounds discovery. A comprehensive understanding of the sponge microbiome and the specific sponge- microbe symbiotic relationships are required to guide the rationale exploration of the unique and untapped microbial resources. This project has developed a novel approach based on Next Generation Sequencing platform to advance our current understanding of sponge microbiome - by comprehensively revealing the composition and structure of the complex microbial communities associated with diverse marine sponges from South Australia. In addition, the hypotheses of sponge microbial specificity at the host sponge order and family levels were tested. The first step in this study was to ascertain the identity of the host sponges to achieve reliable analyses. A new integrated sponge identification protocol (SIP), utilising a multilocus- based molecular protocol in conjunction with the examination of morphological characters, was developed to conduct an effective and reliable sponge classification based on a sample of 37 sponge species. To ensure the commonly used DNA extraction and PCR amplification protocols are efficient with minimal bias toward sponge microbiome analysis, this study has established an appropriate protocol by spiking actinobacterial spores and mycelia into the sponge samples for optimisation. Different DNA yields per unit weight spores and mycelia, and the potential inhibitors in 16S RNA gene PCR amplification were found, highlighting the DNA extraction method validation critical for sponge microbiome analysis. For 16S rRNA gene based metagenomic sequencing, five primer sets targeting different regions were evaluated and validated on 454 pyro- sequencing and Illumina sequencing platforms. The microbial communities for a given sponge species showed substantial differences between the profiles generated by different primer sets. The integrated data demonstrated that the sponge-associated microbial community has the specificity on structure and diversity within a phylogenetic unit, which are valuable in guiding the exploration of the target microbial groups, particularly for the untapped resources. In conclusion, this project developed a pipeline for an unprecedented complete characterisation of the sponge microbiome, which includes reliable identification of sponge samples, efficient extraction of community DNA, PCR optimisation, evaluation of region-specific primer sets for 16S rRNA gene based amplicon sequencing, and bioinformatics analysis, for a rationale guided discovery of untapped marine sponge associated microbial resources.
Keywords: Sponge, Taxonomy, Microbiome, Metagenomic sequencing, Bioinfomatics, Actinobacteria
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Medicine
Supervisor: Chris Franco