Abstract
Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New sequencing technologies produce long reads that can span the entire 16S rRNA gene, but have substantially higher error rates that 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 mixtures of known bacterial strains 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. E. coli strains in the mock communities were correctly classified to the O157:H7 and K12 sub-species clades from the 16S gene sequences recovered by our method. In human fecal samples, our method recovered the full complement of 16S rRNA gene variants in detected E. coli strains and showed strong technical replication.
We discuss the promises and challenges of of classification based on the full complement of multi-copy marker genes such as the 16S rRNA gene. 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.
Author Contributions
BJC designed the research; BJC implemented the algorithm; BJC performed the analysis; BJC wrote the paper; JW, CH and SO developed the amplicon sequencing methodology, performed the amplicon sequencing, and processed the raw sequencing data; CMT, ASG, SKM and MKD collected the human fecal samples.
Footnotes
Conflict of Interest Statement The sequencing data investigated in this manuscript were generated by Pacific Biosciences Inc, Menlo Park, CA. JW, CH and SO are full-time employees at Pacific Biosciences, a company commercializing single-molecule sequencing technologies.