Cargando…
Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing
INTRODUCTION: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10225699/ https://www.ncbi.nlm.nih.gov/pubmed/37256057 http://dx.doi.org/10.3389/fmicb.2023.1179966 |
_version_ | 1785050432272334848 |
---|---|
author | Zhao, Wenxuan Zeng, Wei Pang, Bo Luo, Ming Peng, Yao Xu, Jialiang Kan, Biao Li, Zhenpeng Lu, Xin |
author_facet | Zhao, Wenxuan Zeng, Wei Pang, Bo Luo, Ming Peng, Yao Xu, Jialiang Kan, Biao Li, Zhenpeng Lu, Xin |
author_sort | Zhao, Wenxuan |
collection | PubMed |
description | INTRODUCTION: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time. METHODS: In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%. RESULTS AND DISCUSSION: For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes. |
format | Online Article Text |
id | pubmed-10225699 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-102256992023-05-30 Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing Zhao, Wenxuan Zeng, Wei Pang, Bo Luo, Ming Peng, Yao Xu, Jialiang Kan, Biao Li, Zhenpeng Lu, Xin Front Microbiol Microbiology INTRODUCTION: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time. METHODS: In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%. RESULTS AND DISCUSSION: For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes. Frontiers Media S.A. 2023-05-15 /pmc/articles/PMC10225699/ /pubmed/37256057 http://dx.doi.org/10.3389/fmicb.2023.1179966 Text en Copyright © 2023 Zhao, Zeng, Pang, Luo, Peng, Xu, Kan, Li and Lu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Zhao, Wenxuan Zeng, Wei Pang, Bo Luo, Ming Peng, Yao Xu, Jialiang Kan, Biao Li, Zhenpeng Lu, Xin Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
title | Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
title_full | Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
title_fullStr | Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
title_full_unstemmed | Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
title_short | Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
title_sort | oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10225699/ https://www.ncbi.nlm.nih.gov/pubmed/37256057 http://dx.doi.org/10.3389/fmicb.2023.1179966 |
work_keys_str_mv | AT zhaowenxuan oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT zengwei oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT pangbo oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT luoming oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT pengyao oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT xujialiang oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT kanbiao oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT lizhenpeng oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing AT luxin oxfordnanoporelongreadsequencingenablesthegenerationofcompletebacterialandplasmidgenomeswithoutshortreadsequencing |