Development and application of -omics and bioinformatics approaches for a deeper understanding of infectious diseases systems

Background: Research in infectious diseases underwent a revolution with the uprising of Omics approaches, including, but not limited to, genomics, metagenomics and metatranscriptomics. In fact, there are several examples where Omics approaches showed their potential to tackle different challenges related to the versatile nature of infectious diseases by promoting “studies of one” to “system-wide studies”. In the frame of this PhD programme, we focused on the development and validation of Omics approaches and bioinformatics workflow aiming at tackling mainly diagnostics but also to some extents the treatment of infectious diseases. The four applications presented in this thesis had following specific objectives; (i) to develop and validate a bioinformatics approach aiming at selecting high quality markers among a large amount of complete genomic sequences; (ii) to characterise the viral metagenome of a plant to determine aetiology of a disease that could not be identified and/or fully characterised with other tools; (iii) to assess the potential of metagenomics in the field of personalised medicine and compare its diagnostics accuracy with validated diagnostics tools; and (iv) to make a system-wide survey of microbial populations and estimate its potential to cause harm to humans. Methods: Methodology was specific for each application but as a general rule, we only used published bioinformatics tools that have been used and validated in other studies. This includes, but is not limited to, the BLAST algorithm for the comparison of sequences to various databases and the MIRA assembler to assemble the metagenomics datasets obtained within the different projects. Results: For clarity, the results are summarised by project, corresponding to the different applications investigated during this PhD. Project (i): The developed bioinformatics workflow allowed the selection of highly conserved and specific molecular markers among various viral species with inputs of up to several hundred complete genomic sequences. The quality of the selected markers was successfully validated using several types of molecular assays including real-time PCR, LAMP and Sanger sequencing. Project (ii): We were able to find the aetiology of a grapevine plant presenting leafroll symptoms. A new virus, named Grapevine Leafroll-associated virus 4 Ob, with a thirteen kilobases genome was found in the viral metagenome. Other viruses that were co-identified in the virome were known to be asymptomatic viruses for grapevine, and with the help of additional serological experiences, we were able to confirm that this GLRaV-4 Ob was the causative agent of the Leafroll symptoms. Project (iii): The gut pathobiomes from four patients presenting persistent digestive disorders were fully characterised using a metagenomics approach. Comparison of validated diagnostics tools with this approach showed that the diagnostics rate was in favour of the latter for the detection of bacterial and helminths pathogens and in favour of the validated tools for the detection of viruses and protozoa. Using the same datasets, but compared to a different database, we were also able to screen the stool samples for antimicrobial resistance genes and retrieve potential resistance genes that might interfere with the treatment of these patients. Project (iv): In this project, a system-wide assessment of the microbial communities of the wastewater treatment system was done using a metagenomics approach. We were able to demonstrate how closely the genetic diversity of Escherichia coli and the overall genetic diversity were linked in this environment. We were also able to map the repartition of different pathogenic classes, including bacteria, helminths, intestinal protozoa and viruses as well as to show if and how human waterborne pathogens spread throughout this ecosystem. Conclusion: Omics offer new strategies of how challenges, mainly related to the vast diversity within the research area of infectious diseases, can be tackled. Meta-analyses, like metagenomics or metatranscriptomics are the applications that benefited most from the use of Next-Generation Sequencing technologies, and they now allow system-wide studies where previous studies were only focusing on one parameter (one microbe or one specific gene for instance). However, these Omics approaches have their limitations, mainly due to the bioinformatics challenges they give rise to. As a general conclusion, it is foreseeable that, because of the increased amount of results they generate, Omics approaches, once matured, will be more widely used and will replace standard approaches in the field of infectious diseases