  The high throughput and cost-effectiveness afforded by short-read sequencing
technologies, in principle, enable researchers to perform 16S rRNA profiling of
complex microbial communities at unprecedented depth and resolution. Existing
Illumina sequencing protocols are, however, limited by the fraction of the 16S
rRNA gene that is interrogated and therefore limit the resolution and quality
of the profiling. To address this, we present the design of a novel protocol
for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to
capture more than 90% of sequences in the Greengenes database and with nearly
twice the resolution of existing protocols. Using several in silico and
experimental datasets, we demonstrate that despite the presence of multiple
variable and conserved regions, the resulting shotgun sequences can be used to
accurately quantify the diversity of complex microbial communities. The
reconstruction of a significant fraction of the 16S rRNA gene also enabled high
precision (>90%) in species-level identification thereby opening up potential
application of this approach for clinical microbial characterization.
