Microbial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities in unprecedented detail. This methodology produces long-read sequences including multiple copies of the same complete 16S ribosomal gene and its neighboring internally transcribed spacer (ITS) using rolling-circle amplification. The ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism, the assessment of their relative stoichiometry, and the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to a mock microbial community that was also sequenced using the Illumina platform, demonstrating its accuracy in quantifying the relative abundance and identity of each species. ### Competing Interest Statement The authors have declared no competing interest.
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