Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database.

The increasing number and diversity of genetically modified organisms (GMOs) for the food and feed market calls for the development of advanced methods for their detection and identification. This issue can be addressed by next generation sequencing (NGS). However, the efficiency of NGS-based strategies depends on the availability of bioinformatic methods to find sequences of the transgenic insert and junction regions, which is a challenging topic. To facilitate this task, we have developed Nexplorer, a sequence-based database in which annotated sequences of GM events are stored in a structured, searchable and extractable format. As a proof of concept, we have developed a methodology for the analysis of sequencing data of DNA walking libraries of samples containing GMOs using the database. The efficiency of the method has been tested on datasets representing various scenarios that can be encountered in routine GMO analysis. Database-guided analysis allowed obtaining detailed and reliable information with limited hands-on time. As the database allows for efficient analysis of NGS data, it paves the way for the use of NGS sequencing technology to aid routine detection and identification of GMO.

A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches.

The use of whole-genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), in particular, WGS has the benefit of drastically reducing the time required to generate results compared to most conventional phenotypic methods. Consequently, a multitude of solutions for analyzing WGS MTB data have been developed, but their successful integration in clinical and national reference laboratories is hindered by the requirement for their validation, for which a consensus framework is still largely absent. We developed a bioinformatics workflow for (Illumina) WGS-based routine typing of MTB complex (MTBC) member isolates allowing complete characterization, including (sub)species confirmation and identification (16S, csb/RD, hsp65), single nucleotide polymorphism (SNP)-based antimicrobial resistance (AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome multilocus sequence typing). Workflow performance was validated on a per-assay basis using a collection of 238 in-house-sequenced MTBC isolates, extensively characterized with conventional molecular biology-based approaches supplemented with public data. For SNP-based AMR prediction, results from molecular genotyping methods were supplemented with in silico modified data sets, allowing us to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics of >99% for all assays, except for spoligotyping, where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid in the standardization of bioinformatics tools by the MTB community and other SNP-based applications regardless of the targeted pathogen(s). The bioinformatics workflow is available for academic and nonprofit use through the Galaxy instance of our institute at https://galaxy.sciensano.be.

Phylogenomic Investigation of Increasing Fluoroquinolone Resistance among Belgian Cases of Shigellosis between 2013 and 2018 Indicates Both Travel-Related Imports and Domestic Circulation.

Shigellosis is an acute enteric infection caused mainly by the species Shigella flexneri and Shigella sonnei. Since surveillance of these pathogens indicated an increase in ciprofloxacin-resistant samples collected in Belgium between 2013 and 2018, a subset of 148 samples was analyzed with whole genome sequencing (WGS) to investigate their dispersion and underlying genomic features associated with ciprofloxacin resistance. A comparison between observed phenotypes and WGS-based resistance prediction to ciprofloxacin revealed perfect correspondence for all samples. Core genome multi-locus sequence typing and single nucleotide polymorphism-typing were used for phylogenomic investigation to characterize the spread of these infections within Belgium, supplemented with data from international reference collections to place the Belgian isolates within their global context. For S. flexneri, substantial diversity was observed with ciprofloxacin-resistant isolates assigned to several phylogenetic groups. Besides travel-related imports, several clusters of highly similar Belgian isolates could not be linked directly to international travel suggesting the presence of domestically circulating strains. For S. sonnei, Belgian isolates were all limited to lineage III, and could often be traced back to travel to countries in Asia and Africa, sometimes followed by domestic circulation. For both species, several clusters of isolates obtained exclusively from male patients were observed. Additionally, we illustrated the limitations of conventional serotyping of S. flexneri, which was impacted by serotype switching. This study contributes to a better understanding of the spread of shigellosis within Belgium and internationally, and highlights the added value of WGS for the surveillance of this pathogen.

Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods.

Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a 'push-button' pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.

Validation of a Bioinformatics Workflow for Routine Analysis of Whole-Genome Sequencing Data and Related Challenges for Pathogen Typing in a European National Reference Center: Neisseria meningitidis as a Proof-of-Concept.

Despite being a well-established research method, the use of whole-genome sequencing (WGS) for routine molecular typing and pathogen characterization remains a substantial challenge due to the required bioinformatics resources and/or expertise. Moreover, many national reference laboratories and centers, as well as other laboratories working under a quality system, require extensive validation to demonstrate that employed methods are "fit-for-purpose" and provide high-quality results. A harmonized framework with guidelines for the validation of WGS workflows does currently, however, not exist yet, despite several recent case studies highlighting the urgent need thereof. We present a validation strategy focusing specifically on the exhaustive characterization of the bioinformatics analysis of a WGS workflow designed to replace conventionally employed molecular typing methods for microbial isolates in a representative small-scale laboratory, using the pathogen Neisseria meningitidis as a proof-of-concept. We adapted several classically employed performance metrics specifically toward three different bioinformatics assays: resistance gene characterization (based on the ARG-ANNOT, ResFinder, CARD, and NDARO databases), several commonly employed typing schemas (including, among others, core genome multilocus sequence typing), and serogroup determination. We analyzed a core validation dataset of 67 well-characterized samples typed by means of classical genotypic and/or phenotypic methods that were sequenced in-house, allowing to evaluate repeatability, reproducibility, accuracy, precision, sensitivity, and specificity of the different bioinformatics assays. We also analyzed an extended validation dataset composed of publicly available WGS data for 64 samples by comparing results of the different bioinformatics assays against results obtained from commonly used bioinformatics tools. We demonstrate high performance, with values for all performance metrics >87%, >97%, and >90% for the resistance gene characterization, sequence typing, and serogroup determination assays, respectively, for both validation datasets. Our WGS workflow has been made publicly available as a "push-button" pipeline for Illumina data at https://galaxy.sciensano.be to showcase its implementation for non-profit and/or academic usage. Our validation strategy can be adapted to other WGS workflows for other pathogens of interest and demonstrates the added value and feasibility of employing WGS with the aim of being integrated into routine use in an applied public health setting.