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Next Generation Genome Based High Resolution Tracing of Pathogens

Periodic Report Summary 3 - PATHONGEN-TRACE (Next Generation Genome Based High Resolution Tracing of Pathogens)

Project Context and Objectives:
Next Generation Sequencing (NGS) has fundamentally altered genomic research. The rapid development of this technology has enhanced performance and reduced DNA sequencing costs, widening the spectrum of possible cost-effective applications. The high potential for ultra-fast and accurate molecular typing and diagnostics that NGS promises is being exploited in the medical diagnosis of pathogens. In this context, PathoNGen-Trace (PNGT) aims to develop Next Generation Sequencing and next generation DNA analysis tools into a highly efficient and effective technology for diagnostics and the high-resolution typing of microbial pathogens. An international consortium of leading experts in the field of clinical microbiology, such as the Muenster University, the University of Oxford, and the Research Center Borstel (scientific project coordinator), are working together in the project with well-known commercial enterprises in this area - Applied Maths NV, Genoscreen SAS, Piext BV, and Ridom GmbH.
Three pathogens are being used as models – Mycobacterium tuberculosis complex (Mtbc), methicillin-resistant Staphylococcus aureus (MRSA), and human-pathogenic Campylobacter species (CAMPY). All three pathogens are major causes of human disease worldwide, posing serious medical threats and important challenges for treatment and public health. One third of the world’s population is currently infected with tuberculosis (TB), with new infections occurring at a rate of about one per second globally. Although it remains latent in most cases, over nine million cases of active TB were reported in 2009, with about 1.7 million fatal cases. MRSA is the major cause of hospital-acquired infections, often affecting surgical intensive care units, burn centres, maternity wards and special care baby units. Finally, the pathogens of the genus Campylobacter (C. jejuni and C. coli) are the major causes of bacterial gastroenteritis in humans worldwide and are transmitted from animals to humans especially, but not exclusively, in retail food products. In Europe they are one of the main causes of bacterial intestinal infections, rising above the number of Salmonella infections for the first time in 2005. While classical genotyping has been applied to understand transmission and population structure of these pathogens, recent whole genome sequence (WGS) investigations showed the conventional approaches lack discriminatory power and do not reflect the full level of genome diversity.
The PathoNGen-Trace project aims to overcome drawbacks in conventional typing and diagnosis with NGS-based genotyping for discrimination of clinical isolates at genome wide level. In parallel to the development of new tools, the consortium will also determine yet unknown quantitative parameters of genome evolution on a population basis, in order to calibrate and validate NGS for pathogen genotyping and epidemiological tracing. The main objectives of our project are: (i) to develop new, completely integrated bioinformatics tools for fast and easy quality-controlled data extraction and interpretation for general diagnostics (e.g. drug-resistance) and public health applications (genomics-based molecular epidemiology); (ii) to streamline and implement new internal quality control procedures of the whole NGS process, from sample preparation protocols to final sequence assemblies or mapping; and (iii) to test and validate the performances of NGS for ultra-sensitive/early diagnostics and to monitor the spread of major microbial pathogens.

Project Results:
The major results of the five scientific work packages are:
WP1: Development of Easy-to-use NGS analysis tools and implementation in the software packages SeqSphere+® (Ridom GmbH) and BioNumerics® (Applied Maths NV) was continued. A bacterial core genome definer, a genome wide gene-by-gene typing method, an assembly pipeline and a plain-language reporting system were developed, integrated into the SeqSphere+® and tested for MTBC, MRSA and CAMPY. A command-line free NGS assembly and analysis pipeline, and a high-throughput BioNumerics Calculation Engine (BCE) were implemented in BioNumerics® and tested in a real setting. Standardization and harmonisation of cgMLST approaches discussed in a workshop organized by UOXF and WWU followed by establishment of web based cgMLST nomenclature servers for six different pathogens. The BioNumerics and BNC combination was evaluated by CDC (Atlanta, USA) and is currently routinely used there in surveillance efforts.
Assessment of the sequencing platform, sample preparation and set-up of quality control was performed. This led to the selection of the MiSeq platform and Nextera XT library preparation kits allowing high multiplexing levels. Large strain collections have been sequenced by GSR with FZB, WWU, and UOXF (see below) for analysis in WP3-5. A multiplex PCR based assay coupled to ultra-deep sequencing on a benchtop NGS platform for ultrafast analysis of clinical MTBC strains was developed by GSR in kit format (Deeplex®-MycTB). This is designed for direct application on clinical samples without culture, for highly sensitive species identification, MTBC strain typing and resistance prediction.
To define optimal parameters for WGS of clinical MTBC isolates, NGS platforms and library preparations were evaluated. Illumina chemistry was highly reproducible with optimal performance for MTBC. Transmission analysis parameters were defined by WGS of MTBC strains from longitudinal outbreaks and global collections. Isolates from the Hamburg area (2007 to 2015) were analyzed (1190 isolates in total) to define optimal parameters for WGS based genotyping. Prospective WGS analysis was done for years 2015/2016. Genome data were used to define resistance associated mutations. A genome wide gene-by-gene approach using the Ridom SeqSphere® was developed and implemented.
A comparison of bench-top NGS machines was performed for S. aureus, M. tuberculosis and E. coli. 200 MRSA isolates with epidemiological data were sequenced retrospectively. A population-based study of MRSA isolates from patients in North Rhine-Westphalia was done in a two-month period in 2012. Prospective WGS of all multi-resistant bacteria isolated between 10.2013 and 04.2014 at the University Hospital Münster was done (in total 645 isolates). A web-based visualization and analysis tool called GbGDiv (Gene-by-Gene Diversity) was developed UOXF for analyses of the allelic diversity within a large dataset. By using a high-throughput genome assembly pipeline upstream to GbGDiv, the evolutionary forces in different classes of in total 25,357 S. aureus genomes were investigated.
A high-throughput sequencing pipeline was developed for the generation, assembly, analysis and dissemination of Campylobacter (C. jejuni and C. coli) WGS data and used to characterise large well-defined strain collections. Similarly to WP3/4, a NGS platform comparison was performed. The WGS studies generated new epidemiological datasets and enabled their comparison with other data sets, specifically the on-going campylobacteriosis in Oxfordshire survey. On-going validation of the data from the 2.193 isolates investigated, demonstrate very high levels of accuracy and effectiveness. These data established disease trends over a period of more than a decade in this region, which corresponds to about 1% of the UK total population. The NGS data also enabled the prediction of resistance to luoroquinolone and tetracyclinewith worked with high accuracy.

Potential Impact:
PathoNgen-Trace outcomes will foster the implementation of new and widespread applications of NGS in clinical microbiology and disease surveillance. The NGS kits, methodologies, and software developed for highly effective diagnostics and genotyping of major pathogens have a direct impact on patient management and disease surveillance, and thus reduce health-related costs. Together with the development of bench top WGS and continued reduction of NGS costs, PathoNgen-Trace results will foster a major technological shift towards WGS based approaches.
The development of new tools/technologies will contribute to overcome existing obstacles for large-scale use of NGS by European clinical microbiologists and scientists, thus fostering competitiveness of Europe in biomedical applications. Importantly, the tools have been developed under formats as generic as possible, to be applicable to a wide variety of micro-organisms. In particular, the developed NGS research tools significantly enhance data generation (e.g. better NGS workflow, new technologies) and improve standardisation (ontology, APIs, and kits), quality control (algorithms) and analysis (new bioinformatics tools). This also leads to a much wider application of NGS technology, that will enable the generation of an enormous amount of new knowledge in the European region, that is crucial for increasing the competitiveness of Europe in the areas of "-omics" research and systems biology.
Beyond these new key applications and validation of NGS for medical microbiology, the tools developed will also be usable for other relevant NGS-based applications such as comprehensive microbial genomic characterization for bio-banking, bio-processing and synthetic biology.
The tools and knowledge developed by PathoNGen-Trace will allow for modern molecular surveillance at the outmost ultimate level of resolution by using whole genome data for pathogen tracing. The software solutions develop allow for easy quality-controlled strain classification based on NGS data even for non-specialized users. Due to the development of generic tools and databases, PathoNGen-Trace is likely to continue and extend the success of current typing databases ( SpaServer) and link it to future European surveillance programs from the European Center for Disease Prevention and Control (ECDC).
Development of such new NGS tools for studying population structure/genome evolution of a variety of pathogens by a wide user community has already generated an explosion of knowledge on transmission dynamics, population structure and genome evolution. This will promote improved European disease control measures and a better understanding of virulence and resistance traits of major pathogens. Therefore, the added value for European citizens directly resulting from the project results will be: (i) more comprehensive detection (and therefore better treatment) of relevant pathogen infection parameters at patient level (ii), more specific and sensitive early warning microbial outbreak detection at public health level and therefore more effective combating and prevention of pathogen epidemics (esp. multi-resistant ones). Thus, PathoNGen-Trace is likely to create a significant long lasting "added value" in the European health and bio-industrial research area.

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