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

TL;DR

This paper introduces a novel shotgun Illumina sequencing protocol for 16S rRNA genes that significantly improves resolution and accuracy in profiling complex microbial communities, enabling precise species identification and better diversity quantification.

Contribution

The authors designed a new sequencing protocol that captures over 90% of 16S rRNA sequences and nearly doubles the resolution compared to existing methods.

Findings

Achieves >90% sequence coverage of the Greengenes database

Provides nearly twice the resolution of current protocols

Enables >90% species-level identification accuracy

Abstract

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.