Abstract
BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.
Highlights
Amplifying and sequencing 16S rRNA genes from microbial communities has become a standard technique to survey and compare communities across space, time and environments
Sequencing of near full-length 16S rRNA gene sequences on the Illumina MiSeq We present a method to sequence near full-length 16S rRNA gene amplicons using Illumina technology
Near full-length 16S rRNA gene sequences from an Illumina MiSeq generated by molecular tagging Sequencing of both the full-length and the tagmented amplicon pools was successful with a cluster density of 400–500 k/mm2 and 832,293 read pairs
Summary
Amplifying and sequencing 16S rRNA genes from microbial communities has become a standard technique to survey and compare communities across space, time and environments. High-throughput sequencing methods have made bacterial community profiling routine and affordable This has come at the expense of read length with most platforms covering 250–600 bp of the ∼1,500 bp 16S rRNA gene, where increases in read length are generally accompanied by decreases in read accuracy. Results: We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Conclusions: This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution
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