TY - JOUR
T1 - Validation and Application of Long-Read Whole-Genome Sequencing for Antimicrobial Resistance Gene Detection and Antimicrobial Susceptibility Testing
AU - Weinmaier, Thomas
AU - Conzemius, Rick
AU - Bergman, Yehudit
AU - Lewis, Shawna
AU - Jacobs, Emily B.
AU - Tamma, Pranita D.
AU - Materna, Arne
AU - Weinberger, Johannes
AU - Beisken, Stephan
AU - Simner, Patricia J.
N1 - Funding Information:
Copyright © 2022 Weinmaier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Patricia J. Simner, psimner1@jhmi.edu. The authors declare a conflict of interest. T.W., R.C., A.M., J.W., and S.B. are employees of Ares Genetics GmbH. P.J.S. has received grants and personal fees from Accelerate Diagnostics, OpGen Inc., and BD Diagnostics; grants from bioMérieux, Inc., Affinity Biosensors, and Hardy Diagnostics; and personal fees from Roche Diagnostics, GeneCapture, and Shionogi Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received 7 August 2022 Returned for modification 15 September 2022 Accepted 13 October 2022 Published 19 December 2022
Publisher Copyright:
© 2022 Weinmaier et al.
PY - 2023/1
Y1 - 2023/1
N2 - Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing. We sought to identify best practices in ONT sequencing protocols. As some variability in sequencing results may be introduced by the DNA extraction methodology, we tested three DNA extraction kits across three independent laboratories using a representative set of six bacterial isolates to investigate accuracy and reproducibility of ONT technology. All DNA extraction techniques showed comparable performance; however, the DNeasy PowerSoil Pro kit had the highest sequencing yield. This kit was subsequently applied to 42 sequentially collected bacterial isolates from blood cultures to assess Ares Genetics’s pipelines for predictive whole-genome sequencing antimicrobial susceptibility testing (WGS-AST) performance compared to phenotypic triplicate broth microdilution results. WGS-AST results ranged across the organisms and resulted in an overall categorical agreement of 95% for penicillins, 82.4% for cephalosporins, 76.7% for carbapenems, 86.9% for fluoroquinolones, and 96.2% for aminoglycosides. Very major errors/major errors were 0%/16.7% (penicillins), 11.7%/ 3.6% (cephalosporins), 0%/24.4% (carbapenems), 2.5%/7.7% (fluoroquinolones), and 0%/4.1% (aminoglycosides), respectively. This work showed that, although additional refinements are necessary, ONT sequencing demonstrates potential as a method to perform WGS-AST on cultured isolates for patient care.
AB - Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing. We sought to identify best practices in ONT sequencing protocols. As some variability in sequencing results may be introduced by the DNA extraction methodology, we tested three DNA extraction kits across three independent laboratories using a representative set of six bacterial isolates to investigate accuracy and reproducibility of ONT technology. All DNA extraction techniques showed comparable performance; however, the DNeasy PowerSoil Pro kit had the highest sequencing yield. This kit was subsequently applied to 42 sequentially collected bacterial isolates from blood cultures to assess Ares Genetics’s pipelines for predictive whole-genome sequencing antimicrobial susceptibility testing (WGS-AST) performance compared to phenotypic triplicate broth microdilution results. WGS-AST results ranged across the organisms and resulted in an overall categorical agreement of 95% for penicillins, 82.4% for cephalosporins, 76.7% for carbapenems, 86.9% for fluoroquinolones, and 96.2% for aminoglycosides. Very major errors/major errors were 0%/16.7% (penicillins), 11.7%/ 3.6% (cephalosporins), 0%/24.4% (carbapenems), 2.5%/7.7% (fluoroquinolones), and 0%/4.1% (aminoglycosides), respectively. This work showed that, although additional refinements are necessary, ONT sequencing demonstrates potential as a method to perform WGS-AST on cultured isolates for patient care.
KW - ONT
KW - WGS
KW - WGS-AST
KW - antibiotic resistance
KW - antimicrobial resistance
KW - antimicrobial susceptibility testing
KW - nanopore sequencing
KW - whole-genome sequencing
UR - http://www.scopus.com/inward/record.url?scp=85147048288&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85147048288&partnerID=8YFLogxK
U2 - 10.1128/aac.01072-22
DO - 10.1128/aac.01072-22
M3 - Article
C2 - 36533931
AN - SCOPUS:85147048288
SN - 0066-4804
VL - 67
JO - Antimicrobial agents and chemotherapy
JF - Antimicrobial agents and chemotherapy
IS - 1
ER -