Exploring microbial biodiversity through whole genome analysis using ResolveDNA® Microbiome

Durga M. Arvapalli, Isai Salas-González, Jon S. Zawistowski, Jeff G. Blackinton, Mark Hurt, Gary L. Harton, Jason A.A. West

BioSkryb Genomics, Inc., Durham, NC. Genomic surveillance of microbes is crucial to understand the genetic contributions to host/bacteria interactions. In contrast to metagenomic approaches, whole genome sequencing (WGS) of single bacteria is required to fully reveal the diversity and evolutionary trajectory of the microbiome.

Here, we utilized the ResolveDNA® Microbiome kit to generate high quality genome assemblies from two bacterial species. The workflow utilizes Primary Template-directed Amplification (PTA), a novel technology for low-input and single cell genome amplification with unprecedented coverage uniformity. Gram-negative (E. coli), gram-positive (B. subtilis) single bacterial cells and mixed bacteria (E. coli & B. subtilis) were sorted using fluorescence-activated cell sorting (FACS). The single sorted bacteria were lysed and amplified using the ResolveDNA® Microbiome kit with amplification yields between 50-200 ng and average amplicon size of 1000 bp. ResolveDNA® sequencing libraries were constructed, followed by Illumina MiniSeq sequencing. The reconstructed genome assemblies approximated the empirically estimated genome sizes for each species. Additionally, by employing an unbiased de novo phylogenetic approach we quantitatively validated the high-quality of the assemblies and calculated that the assembly completeness was over 95% for each single bacterium sequenced. Collectively, the data provides evidence that minute numbers of bacteria can be separated, amplified and prepared for next generation analysis (NGS) analysis.

Having successfully analyzed single bacterial genomes, currently we are expanding these findings to computationally deconvolve multi-species bacterial cocktails and to generate datasets from environmental and clinical microbiome samples. We have demonstrated here that ResolveDNA® Microbiome platform is robust to reconstruct high quality genomes for gram-negative and gram-positive bacteria at single-cell resolution, which allows for analysis of complex microbial populations, including those not culturable.