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

Benjamin J Callahan,Casey M Theriot,Sarah K Mcgill,Michael K Dougherty,Joan Wong,Cheryl Heiner,Steve Oh,Ajay S Gulati

doi:10.1093/nar/gkz569

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.

Highlights

The amplification of specific genetic loci by polymerase chain-reaction (PCR) can powerfully focus DNA sequencing on genetic variation of interest
To investigate whether the full complement of 16S rRNA alleles within individual strains could be resolved in more complex natural communities, we focused on all amplicon sequence variants (ASVs) in the fecal samples that were classified as Escherichia coli.In each sample in which an appreciable number of E. colireads was detected, clear bins of ASVs from the same strain could be constructed based on the expected integral ratios between the abundances of intra-genomic alleles, and our knowledge that E. colihas 7 copies of the 16S rRNA gene (Figure 5B)
The potential for species-level classification from full-length 16S rRNA gene amplicon sequencing has been convincingly demonstrated (e.g. Earl 2018), but higher costs, higher error rates and a less-developed ecosystem of computational methods continue to limit the appeal of long-read amplicon sequencing

Summary

Introduction

The amplification of specific genetic loci by polymerase chain-reaction (PCR) can powerfully focus DNA sequencing on genetic variation of interest. Amplicon sequencing effectively detects genetic variation embedded in complex chemical and genetic backgrounds, and is far more cost-effective than untargeted sequencing when large amounts of undesired genetic material is present, as can be the case for host-associated microbial populations or specific genes in large genomes (Franzosa 2015). The precision, sensitivity and low cost of amplicon sequencing have made it a ubiquitous tool utilized in thousands of published scientific studies each year. The genetic loci measured by amplicon sequencing are typically restricted to 100–500 nucleotide regions that fit within the short reads generated by high-throughput sequencing platforms. In studies of functional genes, short reads do not cover even compact viral genes, limiting amplicon sequencing to incomplete measurements of functional genes

Methods

Results

Conclusion