Swee Hoe Ong, Vinutha Uppoor Kukkillaya, Andreas Wilm, Christophe Lay, Eliza Xin Pei Ho, Louie Low, Martin Lloyd Hibberd, Niranjan Nagarajan
(Submitted on 12 Oct 2012)
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.
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This seems like a promising protocol from the perspective of species-level identification, but the obvious issue I see – and it is a major one – is that the protocol requires purification, quantification, fragmentation and barcoding to be performed on a per-sample basis. I would be interested if the authors had a summary of the costs and hands-on time for each step, and then compared this to a traditional fusion amplicon primer approach, perhaps with 96 samples. I appreciate these costs may come down in future and that is mentioned in the discussion, but it is likely to be a sticking point in the near future.
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