Background Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus , Lactobacillus helveticus and Lactobacillus delbrueckii . Results Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats. Conclusions Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities. Electronic supplementary material The online version of this article (10.1186/s12866-019-1500-0) contains supplementary material, which is available to authorized users.

Generating a complete, de novo genome assembly for prokaryotes is often considered a solved problem. However, we here show that Pseudomonas koreensis P19E3 harbors multiple, near identical repeat pairs up to 70 kilobase pairs in length, which contained several genes that may confer fitness advantages to the strain. Its complex genome, which also included a variable shufflon region, could not be de novo assembled with long reads produced by Pacific Biosciences’ technology, but required very long reads from Oxford Nanopore Technologies. Importantly, a repeat analysis, whose results we release for over 9600 prokaryotes, indicated that very complex bacterial genomes represent a general phenomenon beyond Pseudomonas. Roughly 10% of 9331 complete bacterial and a handful of 293 complete archaeal genomes represented this ‘dark matter’ for de novo genome assembly of prokaryotes. Several of these ‘dark matter’ genome assemblies contained repeats far beyond the resolution of the sequencing technology employed and likely contain errors, other genomes were closed employing labor-intense steps like cosmid libraries, primer walking or optical mapping. Using very long sequencing reads in combination with assembly algorithms capable of resolving long, near identical repeats will bring most prokaryotic genomes within reach of fast and complete de novo genome assembly.

Despite the progress made in DNA sequencing over the last decade, reconstructing telomere-to-telomere genome assemblies of large and repeat-rich eukaryotic genomes is still difficult. More accurate basecalls or longer reads could address this issue, but no current sequencing platform can provide both simultaneously. Perennial ryegrass (Lolium perenne L.) is an example of an important species for which the lack of a reference genome assembly hindered a swift adoption of genomics-based methods into breeding programs. To fill this gap, we optimized the Oxford Nanopore Technologies sequencing protocol, obtaining sequencing reads with a N50 of 62 kb—an unprecedented value for a plant sample. The assembly of such reads produced a highly complete (2.3 of 2.7 Gb), correct (QV 45), and contiguous (contig N50 and N90 11.74 and 3.34 Mb, respectively) genome assembly. We show how read length was key in determining the assembly contiguity. Sequence annotation revealed the dominance of transposable elements and repeated sequences (81.6% of the assembly) and identified 38,868 protein coding genes. Almost 90% of the bases could be anchored to seven pseudomolecules, providing the first high quality haploid reference assembly for perennial ryegrass. This protocol will enable producing longer Oxford Nanopore Technology reads for more plant samples and ushering forage grasses into modern genomics-assisted breeding programs.

Background The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. Results We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. Conclusions We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract.