Schlögl, Johannes (2021) Targeted enrichment of sponge-associated microbes using fixation-free Fluorescence in-situ hybridization (FISH) and cell sorting. (Bachelor thesis), Julius-Maximilians-Universität Würzburg, Würzburg, Germany, 63 pp.

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Abstract

The emergence and improvement of novel molecular, culture-independent techniques and bioinformatics revolutionized the field of microbiology at an unseen speed and deliver insights into the so-called microbial dark matter. Especially through Next-Generation-Sequencing (NGS) methods and bioinformatics, enormous metagenomic data was and is delivered to diverse commonly used sequence databases. But high-throughput sequencing or even single-cell genomics are usually untargeted, and a random selection of microorganisms is sequenced hampering the access to low abundant microorganisms especially in complex environments. Only recently, the targeted separation of low abundant microbes through the combination of the established methods fixation-free Fluorescence in-situ Hybridization (ff-FISH) and Fluorescence activated cell sorting (FACS) using the 16S rRNA gene sequence as phylogenetic marker was described by Dam et al. (2020). This approach allows to specifically detect and enrich taxonomically defined clades of microorganisms for genomic analysis and could open a new research field with promising results. In this study I applied ff-FISH to specifically detect members of the microbiome of two marine sponges: Candidatus Halichondribacter symbioticus, first described by Knobloch et al. (2019), from the Baltic Sea sponge Halichondria panicea; and bacteria affiliated with Anaerolineae, Caldilineae, and SAR202 from the microbiome of the Mediterranean sponge Aplysina aerophoba. As controls the method was tested on two culturable bacteria, Escherichia coli K12 and an isolate from H. panicea affiliated with Ca. H. symbioticus called Hal240. After successful method establishment the labelled microorganisms were analysed using FACS. I showed that ff-FISH is a feasible, culture-independent method for a targeted enrichment of microbes of interest but is also challenging and might need improvement and establishment to account for different cell characteristics of diverse microbial phylogenetic groups.

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