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Volume 6, Issue 3

nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples Open Access

Abstract

Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): dual-barcode multiplexing , meticillin-resistant Staphylococcus aureus (MRSA) , MinION , molecular typing , multilocus sequence typing (MLST) and nanopore
Funding
This study was supported by the:
  • Ministry of Science and Technology, Taiwan (Award MOST 106-2923-B-400-001-MY3)
    • Principle Award Recipient: Yu-Chieh Liao
  • National Health Research Institutes (Award PH-107-PP-05)
    • Principle Award Recipient: Yu-Chieh Liao
  • National Health Research Institutes (Award IV-107-PP-07)
    • Principle Award Recipient: Feng-Jui Chen
© 2020 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
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2020-02-17
2024-04-25
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nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples
M Gen 6, e000336 (2020); https://doi.org/10.1099/mgen.0.000336
/content/journal/mgen/10.1099/mgen.0.000336
/content/journal/mgen/10.1099/mgen.0.000336
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