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The high throughput and cost-effectiveness afforded by short-read sequencingtechnologies, in principle, enable researchers to perform 16S rRNA profiling ofcomplex microbial communities at unprecedented depth and resolution. ExistingIllumina sequencing protocols are, however, limited by the fraction of the 16SrRNA gene that is interrogated and therefore limit the resolution and qualityof the profiling. To address this, we present the design of a novel protocolfor shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized tocapture more than 90% of sequences in the Greengenes database and with nearlytwice the resolution of existing protocols. Using several in silico andexperimental datasets, we demonstrate that despite the presence of multiplevariable and conserved regions, the resulting shotgun sequences can be used toaccurately quantify the diversity of complex microbial communities. Thereconstruction of a significant fraction of the 16S rRNA gene also enabled highprecision (>90%) in species-level identification thereby opening up potentialapplication of this approach for clinical microbial characterization.

Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences