RT Journal Article
SR Electronic
T1 High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 392332
DO 10.1101/392332
A1 Benjamin J Callahan
A1 Joan Wong
A1 Cheryl Heiner
A1 Steve Oh
A1 Casey M Theriot
A1 Ajay S Gulati
A1 Sarah K McGill
A1 Michael K Dougherty
YR 2019
UL http://biorxiv.org/content/early/2019/05/20/392332.abstract
AB 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.