Rooney, A. M. and Raphenya, A. R. and Melano, R. G. and Seah, C. and Yee, N. R. and MacFadden, D. R. and McArthur, A. G. and Schneeberger, P. H. H. and Coburn, B.. (2022) Performance characteristics of next-generation sequencing for the detection of antimicrobial resistance determinants in Escherichia coli genomes and metagenomes. mSystems, 7 (3). e0002222.
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Abstract
Short-read sequencing can provide detection of multiple genomic determinants of antimicrobial resistance from single bacterial genomes and metagenomic samples. Despite its increasing application in human, animal, and environmental microbiology, including human clinical trials, the performance of short-read Illumina sequencing for antimicrobial resistance gene (ARG) detection, including resistance-conferring single nucleotide polymorphisms (SNPs), has not been systematically characterized. Using paired-end 2 x 150 bp (base pair) Illumina sequencing and an assembly-based method for ARG prediction, we determined sensitivity, positive predictive value (PPV), and sequencing depths required for ARG detection in an Escherichia coli isolate of sequence type (ST) 38 spiked into a synthetic microbial community at varying abundances. Approximately 300,000 reads or 15x genome coverage was sufficient to detect ARGs in E. coli ST38, with comparable sensitivity and PPV to ~100x genome coverage. Using metagenome assembly of mixed microbial communities, ARG detection at E. coli relative abundances of 1% would require assembly of approximately 30 million reads to achieve 15x target coverage. The minimum sequencing depths were validated using public data sets of 948 E. coli genomes and 10 metagenomic rectal swab samples. A read-based approach using k-mer alignment (KMA) for ARG prediction did not substantially improve minimum sequencing depths for ARG detection compared to assembly of the E. coli ST38 genome or the combined metagenomic samples. Analysis of sequencing depths from recent studies assessing ARG content in metagenomic samples demonstrated that sequencing depths had a median estimated detection frequency of 84% (interquartile range: 30%-92%) for a relative abundance of 1%. IMPORTANCE Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15x coverage for ARG detection for E. coli in metagenomes. We demonstrate that for low relative abundances, sequencing depths of ~30 million reads or more may be required for adequate sensitivity for many applications.
Faculties and Departments: | 09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) 09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology (MPI) > Helminth Drug Development (Keiser) |
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UniBasel Contributors: | Schneeberger, Pierre |
Item Type: | Article, refereed |
Article Subtype: | Research Article |
ISSN: | 2379-5077 (Print)2379-5077 (Linking) |
Note: | Publication type according to Uni Basel Research Database: Journal article |
Language: | English |
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edoc DOI: | |
Last Modified: | 27 Dec 2022 21:17 |
Deposited On: | 27 Dec 2022 21:17 |
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