The decreasing costs in genomic sequencing over the past decade have inspired researchers to apply shotgun next-generation sequencing to entire microbial communities. While the reads generated typically cannot be assembled cleanly into individual genomes, there is often enough information produced to identify most microbes present in the population. However, this approach lacks sufficient resolution to link taxonomy with function.
Applications ranging from clinical microbiome analysis to environmental metagenomics require the production of high-quality genome assemblies for each microbial member of a community. With the goal of yielding such information from metagenomics experiments, Ami Bhatt and collaborators at Stanford University developed a method that uses a new assembler alongside a 10x Genomics long-range workflow that includes automated DNA size selection. In this bioRxiv preprint, the authors report that their novel technique enables successful sequencing of human and marine microbiome samples.
The assembler, named Athena, can produce high-quality, de novo individual draft genomes from microbial communities. It works by analyzing the “read clouds” produced by 10x Genomics technology, which links short sequencing reads to provide long-range genomic data.
Bhatt and her colleagues first tested the ability of the approach to assemble a mock microbial community, and then evaluated samples collected from the human gut and the sea floor. In the latter sample types, the Athena-powered method was able to generate contiguous assemblies of individual microbes present as compared to short read and SLR assembly. Indeed, Bhatt et al. report that their “approach combines the advantages of both short read and [synthetic long read] approaches, and is capable of producing many highly contiguous drafts (>200kb N50, <10 contigs) with as little as 20x raw short-read coverage.” This is particularly significant in the case of the marine sediment community, as this type of sample is significantly more microbially complex than a human stool sample.
The workflow includes DNA size-selection via the BluePippin instrument as a key part of the sample prep process for the 10x Genomics Chromium platform used for library sequencing. This process ensures optimal results from the long-range technology, allowing it to focus on the large DNA fragments that will generate the most useful information from the linked short reads.
Bhatt et al. note that their pipeline is cost-effective and will allow scientists to perform experiments “at a price point that gives it relevance to the broader microbiome community.” Importantly, they conclude that their novel “approach will be a significant step forward in enabling comparative genomics for bacteria, enabling fine-grained inspection of microbial evolution within complex communities.”Image Credit: Frontierofficial