Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing

Noelle R Noyes,Paul S Morley,Steven M Lakin,Chris J Dean,Pablo Rovira,Kenneth L Jones,Jennifer N Martin,Zaid Abdo,Jennifer K Parker,Jaime Ruiz,Christina A Boucher,Keith E Belk,Maggie E Weinroth,Robert A Raymond,Enrique Doster

doi:10.1186/s40168-017-0361-8

Noelle R Noyes, Paul S Morley + Show 13 more

Open Access

https://doi.org/10.1186/s40168-017-0361-8

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Abstract

BackgroundShotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for < 1% of all sequenced DNA, leading to limitations in detection of low-abundance resistome-virulome elements. This study describes the extent and composition of the low-abundance portion of the resistome-virulome, using a bait-capture and enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias.ResultsThe use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins.ConclusionsThese results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

Highlights

Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis
Bait-capture and enrichment enabled access to > 1000 additional, low-abundance gene sequences Across all 64 sequenced libraries, we identified 2490 unique antimicrobial resistance (AR), metal resistance (MR), biocide resistance (BR), and virulence factor (VF) gene accessions across 48 unique classes of resistance and virulence
The majority of these additional 1441 genes originated from AR, BR, and MR genes (1155/1441 or 80.2%), and the majority of these were specific to AR (999/1155 or 86.5%, Additional file 2: File S1)

Summary

Introduction

Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. In previously published fecal metagenomic datasets sampled to a depth of ~ 100 M reads, fewer than 100,000 reads are typically attributed to the resistome [10, 11], meaning that > 99% of sequences could be considered “off-target” if the resistome is the primary study interest. This is of particular concern for epidemiological AMR surveillance efforts, which aim to detect AMR genes in large numbers of continuously collected samples and cannot tolerate cost inefficiencies. Very little has been described regarding the low-abundance portion of the resistome, perhaps because it is difficult to access

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