Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel

Expansion of the Genomic Encyclopedia of Bacteria and Archaea

To date the vast majority of bacterial and archaeal genomes sequenced are of rather limited phylogenetic diversity as they were chosen based on their physiology and/ or medical importance. The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project (Wu et al. 2009) is aimed at systematically filling the gaps of the tree of life with phylogenetically diverse reference genomes. However more than 99 percent of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes of these largely mysterious species. These limitations gave rise to the GEBA uncultured project. Here we propose to use single cell genomics to massively expand the Genomic Encyclopedia of Bacteria and Archaea by targeting 80 single cell representatives of uncultured candidate phyla which have no or very few cultured representatives. Generating these reference genomes of uncultured microbes will dramatically increase the discovery rate of novel protein families and biological functions, shed light on the numerous underrepresented phyla that likely play important roles in the environment, and will assist in improving the reconstruction of the evolutionary history of Bacteria and Archaea. Moreover, these data will improve our ability to interpret metagenomics sequence data from diverse environments, which will be of tremendous value for microbial ecology and evolutionary studies to come

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

[Background] Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva.[Results] We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously.[Conclusions] Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.The work (proposal: 10.46936/10.25585/60000940) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. RS, MB, JL, and TW are supported by NIH grant R01AI168993. The John Templeton Foundation (grant nos. 51250 and 60973) supported TT and SVD, and the Gordon and Betty Moore Foundation grants (GBMF7617 and GBMF9340) supported SVD. MD is supported by the Generalitat Valenciana program GenT grant number CDEIGENT/2021/008. SPE is supported by a FPU grant from the Spanish Ministry of Universities (Reference: FPU20/05756).Peer reviewe

Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

BackgroundNudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva.ResultsWe recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously.ConclusionsAltogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract