Charting and deconvolution of microbial diversity by high-quality single-cell genomic reconstruction utilizing ResolveDNA Microbiome

I. Salas-González, T. Morozova, A. Snediker, D. Arvapalli, J. Zawistowski, J. Blackinton, J. West

BioSkryb Genomics Inc, Durham, NC

The causes and consequences of microbial variation across biomes have been the subject of intense study for over a century. Specifically, bacterial niche partitioning has been shown to play fundamental roles in the structuring and maintenance of global biogeochemical cycles. Additionally, eukaryotic organisms are inhabited by complex microbial communities, collectively named the microbiota, that have been shown to modulate diverse host physiological traits ultimately impacting the host fitness. A bottleneck to unraveling the vast uncharted bacterial diversity across biomes has been the lack of robust technology to faithfully amplify the femtogram quantities of DNA of a single bacterium. Here we present ResolveDNA Microbiome, a whole-genome amplification technology, to reconstruct high-quality genomes of single bacteria.

We benchmarked the ability of our protocol to yield high quality reconstructed bacterial genomes at different levels of community complexity, both at the phylogenetic and cell quantity levels, by performing whole genome sequencing of a FACS-sorted co-culture experiment between the gram-positive bacteria B. subtillis and the gram-negative bacteria E. coli. Following sorting, library preparation and sequencing, we filtered low quality reads and performed de novo bacterial assembly followed up by genome deconvolution using differential depth coverage across the assembly contigs. Evaluation of the quality of the assemblies was performed using an unbiased phylogenetic single-copy marker approach that validated that the reconstructed assemblies in our experiment had levels of completeness over 95% and levels of contamination lower than 1%. Finally, we developed a novel computational pipeline, that leverages random fragments of contaminant DNA within an amplified reaction, to estimate that the high-quality assemblies from our experiment were derived from samples with different levels of cells within each reaction, ranging from 1 cell up to 5 cells. The data presented here demonstrates that ResolveDNA Microbiome can be used to assemble bacterial genomes, at different levels of phylogenetic divergence and cell quantity, with high reliability thus permitting to deconvolute microbial communities into their original constitutes with unseen quality.