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Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. These platforms have allowed researchers to significantly improve the design of their experiments. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The synthetic mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 2.16% to 0.32%. Unfortunately, this error rate was still 16-times higher than the error rate that has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the longer reads frequently provided better classification, the wider adoption of this approach for 16S rRNA gene sequencing is likely limited by its high sequencing error and low yield of sequencing data relative to the other available platforms.
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