Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates.
Base Sequence
DNA, Bacterial
/ genetics
Disease Outbreaks
Drug Resistance, Bacterial
/ genetics
Escherichia coli Infections
/ epidemiology
Escherichia coli O157
/ genetics
Feces
/ microbiology
Humans
Multilocus Sequence Typing
Phylogeny
Plasmids
/ genetics
Polymorphism, Single Nucleotide
Red Meat
/ microbiology
Serogroup
Virulence
/ genetics
Whole Genome Sequencing
/ methods
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
04 09 2020
04 09 2020
Historique:
received:
23
12
2019
accepted:
11
08
2020
entrez:
5
9
2020
pubmed:
6
9
2020
medline:
20
3
2021
Statut:
epublish
Résumé
Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.
Identifiants
pubmed: 32887913
doi: 10.1038/s41598-020-71207-3
pii: 10.1038/s41598-020-71207-3
pmc: PMC7474065
doi:
Substances chimiques
DNA, Bacterial
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
14649Références
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