Abstract
BackgroundComplete 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.ResultsTwo 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.ConclusionsHere, 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.
Highlights
Complete and contiguous genome assemblies greatly improve the quality of subsequent systemswide functional profiling studies and the ability to gain novel biological insights
We observed that the prophage inserted just upstream of a Discussion In this pilot study, we demonstrated the feasibility of complete de novo genome assembly of all dominant species directly from low-complexity metagenomes using third generation long-read sequencing
Relying on long reads from third generation sequencing technologies, we demonstrate the feasibility of de novo assembling finished metagenome-assembled genomes (MAGs) for the dominant strains from cheese starter cultures, which represent low-complexity metagenomes
Summary
Complete and contiguous genome assemblies greatly improve the quality of subsequent systemswide functional profiling studies and the ability to gain novel biological insights. Targeted metagenomic approaches, including the analysis of variable regions of the 16S rRNA, have been widely used to describe the composition of microbial communities [1] They are useful when a high throughput of samples, deep sequencing of the chosen marker genes and the detection of low-abundance taxa is required. For a higher resolution assessment of the entire functional potential of microbial communities, whole metagenome shotgun (WMGS) sequencing approaches provide important advantages. They allow researchers to go beyond sequencing and classifying individual genes of species by covering plasmids, prophages and lytic phages [2, 3], which harbor additional functions and play important roles in shaping microbial communities. Through the analysis of methylation profiles, one can link extrachromosomal genetic elements (e.g., plasmids) to their respective host species [4, 5]
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