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Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

ABSTRACT: Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.

SUBMITTER: Driscoll CB

PROVIDER: S-EPMC5248499 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Driscoll Connor B CB Otten Timothy G TG Brown Nathan M NM Dreher Theo W TW

Standards in genomic sciences 20170119

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hy ...[more]

PMID: 28127419

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Project description:BackgroundLong-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample.ResultsWe used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific -Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345-, whereas others are more broadly distributed among animal and human microbiomes -Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to detect mobilome functions, usually missed in short-read MAGs.ConclusionsWe recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertion and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

Dataset Information

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Publications

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

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