Culture-independent methods and high-throughput sequencing applied to evolutionary microbial genomics

Abstract: Culture-independent methods allow us to better understand the diversity of microorganisms on earth. By omitting the culturing step, we can gain access to species previously out of our reach.To better capture and understand the diversity of microorganisms, we have developed an alternative to 16S amplicon sequencing. This method exploits the fact that the rRNA operon in most prokaryotes contains both the 16S and the 23S genes in a determined order, close to each other. Combining this with PacBio long molecule sequencing, we can now generate amplicon containing the 16S-ITS-23S operon, allowing for a stronger phylogenetic signal.Using two culture-independent methods, single-cell genomics and metagenomics, we have been able to fully sequence the genome of two archaeal endosymbionts from the genera Methanobrevibacter and Methanocorpusculum. These methanogenic archaea were isolated from the ciliates Nyctotherus ovalis and Metopus contortus. The genomic data show evidence of genome degradation, mainly through pseudogenization of genes. These genomes represent the first genomic data from archaeal endosymbionts.The endosymbiotic archaea we have sequenced live in close proximity to hydrogen producing mitochondria, or hydrogenosomes. In ciliates it has previously been shown that the hydrogenosome of N. ovalis has retained a genome, something many other ciliate hydrogenosome have not. Using genomic and transcriptomic data from seven anaerobic ciliates, we have been able to show that these ciliate have independently evolved from ancestral mitochondria, and show various degrees of genome reduction.Furthermore we have been able to shed some new light on haloarchaeal evolution by reconstructing the genomes of five species belonging to the Marine Group IV archaea. These archaea have previously been found to be closely related to the haloarchaea, however they are not halophiles. All five genomes were obtained using metagenomic binning of the publicly available TARA Oceans dataset. The genomes are all of high quality and completeness, and are used in tree-aware ancestral reconstruction, in order to try to better understand the evolutionary transition from an anaerobic methanogen to a halophile.These studies all show how culture-independent method are powerful tools in gathering genomic information about microbes we are currently unable to culture in the lab.