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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

View ORCID ProfileBenjamin J Callahan, Joan Wong, Cheryl Heiner, Steve Oh, Casey M Theriot, Ajay S Gulati, Sarah K McGill, Michael K Dougherty
doi: https://doi.org/10.1101/392332
Benjamin J Callahan
1Department of Population Health & Pathobiology, North Carolina State University, Raleigh, NC 27607
2Bioinformatics Research Center, North Carolina State University, Raleigh, NC 27695
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  • ORCID record for Benjamin J Callahan
  • For correspondence: benjamin.j.callahan@gmail.com
Joan Wong
3Pacific Biosciences of California, Inc., Menlo Park, CA 94025
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Cheryl Heiner
3Pacific Biosciences of California, Inc., Menlo Park, CA 94025
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Steve Oh
3Pacific Biosciences of California, Inc., Menlo Park, CA 94025
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Casey M Theriot
1Department of Population Health & Pathobiology, North Carolina State University, Raleigh, NC 27607
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Ajay S Gulati
4Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
5Department of Pediatrics, Division of Gastroenterology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
6Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Sarah K McGill
7Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Michael K Dougherty
7Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Abstract

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate.

In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed E. coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains.

There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted May 20, 2019.
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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution
Benjamin J Callahan, Joan Wong, Cheryl Heiner, Steve Oh, Casey M Theriot, Ajay S Gulati, Sarah K McGill, Michael K Dougherty
bioRxiv 392332; doi: https://doi.org/10.1101/392332
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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution
Benjamin J Callahan, Joan Wong, Cheryl Heiner, Steve Oh, Casey M Theriot, Ajay S Gulati, Sarah K McGill, Michael K Dougherty
bioRxiv 392332; doi: https://doi.org/10.1101/392332

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