Transversal Activities in Applied Genomics, Sciensano, Brussels, Belgium.
Department of Information Technology, IDLab, Ghent University, IMEC, Ghent, Belgium.
Sci Rep. 2020 Sep 4;10(1):14649. doi: 10.1038/s41598-020-71207-3.
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.
全基因组测序（WGS）已被证明是细菌分离物特征描述和相关性确定的终极工具。然而，需要标准化和协调的工作流程，例如 DNA 提取，以确保稳健和可交换的 WGS 数据。（国际）实验室之间的数据共享对于支持食源性致病菌控制至关重要，包括暴发调查。本研究评估了八种商业 DNA 制备试剂盒对以下方面的潜在影响：（i）用于 Nextera XT 文库制备的 DNA 质量；（ii）MiSeq 测序（数据质量、对质粒和染色体参考的读映射）；和（iii）WGS 数据分析，即分离物特征描述（血清型、毒力和抗微生物药物耐药性基因分型）和系统发育相关性（核心基因组多位点序列分型和单核苷酸多态性分析）。产志贺毒素大肠杆菌（STEC）被选为案例研究。总体而言，DNA 提取试剂盒的选择不会影响数据质量和分离物之间推断的系统发育关系，无论 DNA 溶液缓冲液中是否存在 EDTA 等混杂因素。然而，STEC 特征描述的完整性虽然没有实质性影响，但受到试剂盒质粒提取性能的影响，尤其是在使用 Nextera XT 文库制备时。本研究有助于解决 WGS 标准化协议的挑战，以支持数据可移植性，并充分发挥其潜力。
