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- Alneberg, Johannes, et al.
(author)
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Genomes from uncultivated prokaryotes: a comparison of metagenome-assembled and single-amplified genomes
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Other publication (other academic/artistic)abstract
- Background: Prokaryotes dominate the biosphere and regulate biogeochemical processes essential to all life. Yet, our knowledge about their biology is for the most part limited to the minority that has been successfully cultured. Molecular techniques now allow for obtaining genome sequences of uncultivated prokaryotic taxa, facilitating in-depth analyses that may ultimately improve our understanding of these key organisms.Results: We compared results from two culture-independent strategies for recovering bacterial genomes: single-amplified genomes and metagenome-assembled genomes. Single-amplified genomes were obtained from samples collected at an offshore station in the Baltic Sea Proper and compared to previously obtained metagenome-assembled genomes from a time series at the same station. Among 16 single-amplified genomes analyzed, seven were found to match metagenome-assembled genomes, affiliated with a diverse set of taxa. Notably, genome pairs between the two approaches were nearly identical (>98.7% identity) across overlapping regions (30-80% of each genome). Within matching pairs, the single-amplified genomes were consistently smaller and less complete, whereas the genetic functional profiles were maintained. For the metagenome-assembled genomes, only on average 3.6% of the bases were estimated to be missing from the genomes due to wrongly binned contigs; the metagenome assembly was found to cause incompleteness to a higher degree than the binning procedure.Conclusions: The strong agreement between the single-amplified and metagenome-assembled genomes emphasizes that both methods generate accurate genome information from uncultivated bacteria. Importantly, this implies that the research questions and the available resources are allowed to determine the selection of genomics approach for microbiome studies.
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- Lind, Anders E., 1983-
(author)
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Culture-independent methods and high-throughput sequencing applied to evolutionary microbial genomics
- 2018
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Doctoral thesis (other academic/artistic)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.
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