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Nucleic Acids Res. 2019 Jul 3. pii: gkz569. doi: 10.1093/nar/gkz569. [Epub ahead of print]

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

Author information

1
Department of Population Health & Pathobiology, North Carolina State University, Raleigh, NC 27607, USA.
2
Bioinformatics Research Center, North Carolina State University, Raleigh, NC 27695, USA.
3
Pacific Biosciences of California, Inc., Menlo Park, CA 94025, USA.
4
Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
5
Department of Pediatrics, Division of Gastroenterology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
6
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
7
Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

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 Escherichia 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.

PMID:
31269198
DOI:
10.1093/nar/gkz569

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