Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system
A peer-reviewed article of this Preprint also exists.
Author and article information
Abstract
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. The result of the added sequencing depth has been significant improvements in experimental design. 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-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The 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 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.
Cite this as
2016. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system. PeerJ PrePrints 4:e778v2 https://doi.org/10.7287/peerj.preprints.778v2note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
Author comment
We updated the underlying data by resequencing the samples using the P6-C4 PacBio chemistry. The approach is similar to the previous version, but the overall conclusion has changed significantly.
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Additional Information
Competing Interests
Sarah K Highlander is an employee of JCVI.
Author Contributions
Patrick D Schloss conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Matthew L Jenior performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Charles C. Koumpouras performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Sarah L Westcott analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Sarah K Highlander conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Human Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
The University of Michigan Institutional Review Board (HUM00057066)
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
University of Michigan
Committee on Use and Care of Animals (PRO00004877)
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The raw data can be obtained from the Sequence Read Archive at NCBI under accession SRP051686, which are associated with BioProject PRJNA271568.
Data Deposition
The following information was supplied regarding data availability:
Detailed methods including this paper as an R markdown file are available as a public
online repository (https://github.com/SchlossLab/Schloss_PacBio16S_PeerJ_2016).
Funding
This study was supported by grants from the NIH (R01HG005975, R01GM099514 and P30DK034933 to PDS and U54HG004973 to SKH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
