Transversal activities in Applied Genomics, Sciensano, Brussels (1050), Belgium.
Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent (9000), Belgium.
Microb Genom. 2021 Nov;7(11). doi: 10.1099/mgen.0.000699.
Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing, providing a resolution unattainable with conventional molecular methods. Data generated with Illumina sequencers can however only be analysed after the sequencing run has finished, thereby losing valuable time during emergency situations. We evaluated both the effect of decreasing overall run time, and also a protocol to transfer and convert intermediary files generated by Illumina sequencers enabling real-time data analysis for multiple samples part of the same ongoing sequencing run, as soon as the forward reads have been sequenced. To facilitate implementation for laboratories operating under strict quality systems, extensive validation of several bioinformatics assays (16S rRNA species confirmation, gene detection against virulence factor and antimicrobial resistance databases, SNP-based antimicrobial resistance detection, serotype determination, and core genome multilocus sequence typing) for three bacterial pathogens (, , and Shiga-toxin producing ) was performed by evaluating performance in function of the two most critical sequencing parameters, i.e. read length and coverage. For the majority of evaluated bioinformatics assays, actionable results could be obtained between 14 and 22 h of sequencing, decreasing the overall sequencing-to-results time by more than half. This study aids in reducing the turn-around time of WGS analysis by facilitating a faster response in time-critical scenarios and provides recommendations for time-optimized WGS with respect to required read length and coverage to achieve a minimum level of performance for the considered bioinformatics assay(s), which can also be used to maximize the cost-effectiveness of routine surveillance sequencing when response time is not essential.
全基因组测序（WGS）已成为细菌爆发调查和病原体分型的参考标准，为常规分子方法无法达到的分辨率提供了支持。然而，使用 Illumina 测序仪生成的数据只能在测序运行完成后进行分析，从而在紧急情况下浪费了宝贵的时间。我们评估了缩短总运行时间的效果，以及一种协议，该协议可以传输和转换 Illumina 测序仪生成的中间文件，以便在正向读取测序后，对同一正在进行的测序运行中的多个样本进行实时数据分析。为了便于在严格质量体系下运行的实验室实施，我们对三个细菌病原体（、和产志贺毒素）的多个生物信息学检测（16S rRNA 物种确认、针对毒力因子和抗生素耐药性数据库的基因检测、基于 SNP 的抗生素耐药性检测、血清型确定和核心基因组多位点序列分型）进行了广泛验证，评估了两个最关键的测序参数（读长和覆盖率）的性能。对于大多数评估的生物信息学检测，在测序 14 到 22 小时之间可以获得可操作的结果，将总测序到结果的时间缩短了一半以上。本研究通过为时间关键型场景提供更快的响应，有助于缩短 WGS 分析的周转时间，并针对所需的读长和覆盖率提供 WGS 的时间优化建议，以实现考虑的生物信息学检测的最低性能水平，这也可用于在响应时间不是关键因素时最大化常规监测测序的成本效益。
