Abstract
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.
Highlights
Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination
A high variety of commercial DNA extraction kits is being used for WGS of foodborne pathogens[11,12,13,14], the literature evaluating the potential impact of DNA extraction workflows on WGS and subsequent isolate characterization and phylogeny is rather limited
The isolates originated from different matrices, and were previously conventionally serotyped as O157:H7 containing the pO157 p lasmid[19], and O113:H21 containing the pO113 plasmid[20]
Summary
Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. 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). A high variety of commercial DNA extraction kits is being used for WGS of foodborne pathogens[11,12,13,14], the literature evaluating the potential impact of DNA extraction workflows on WGS and subsequent isolate characterization and phylogeny is rather limited These studies have demonstrated that DNA extraction kit choice does not influence downstream WGS data analysis of different Gram-negative and -positive pathogens[15,16], except for salting-out methods that can deplete small plasmids (< 5 kb)[16]. DNA extraction kit components containing confounding factors such as ethylenediaminetetraacetic acid (EDTA) can diminish or inhibit enzyme activation[17] required for library preparation and sequencing
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