Development of novel bioinformatic pipelines for MinION-based DNA barcoding

DNA-barcoding is the process of taxonomic identification based on the sequence of a marker gene. When complex samples are analysed, we refer in particular to meta-barcoding. Barcoding has traditionally been performed with Sanger sequencing platform. The emergence of second-generation sequencing platforms, mainly represented by Illumina, enabled the high-throughput sequencing of hundreds of samples, and allowed the characterization of complex samples through meta-barcoding experiments. However, fragments sequenced with the Illumina platform are shorter than 600 bp, and this greatly limits taxonomic resolution of closely related species. Moreover, both these platforms suffer of long turnaround time, since they require shipping the samples to a sequencing facility, and complex regulations may hamper the export of material out of the country of origin. More recently, Oxford Nanopore Technologies provided the MinION, a portable and cheap third-generation sequencer, which has the potential of overcoming issues of currently available platforms, thanks to the production of long sequencing reads. However, MinION reads suffer of high error rate, therefore suitable analysis pipelines are needed to overcome this issue. In this thesis I describe the development of bioinformatic pipelines for MinION-based DNA barcoding. Starting from the analysis of single samples, I show how improvements both in sequencing chemistry and in software now allow obtaining consensus sequences directly in the field, with accuracy comparable with Sanger. Conversely, when analysing complex samples, sequencing reads cannot be collapsed for reducing the error rate. However, bioinformatic approaches exploiting increased read length largely compensate the higher error rate, resulting in high correlation between MinION and Illumina up to genus level, and a more marked sensitivity of MinION platform to detect spiked-in indicator species. In conclusion, the results presented in this thesis show that bioinformatic pipelines for the analysis of MinION reads can largely mitigate platform issues, paving the way for this platform to become the gold-standard for barcoding in the near future

Comparison of an Oxford Nanopore Technologies Sequencing Platform to Existing Sequencing Methods for Differential Expression Studies

As the genomics revolution continues, there is constant pressure to make sequencing technology more accessible and practical for a growing series of applications. Existing sequencing technologies are often prohibitively expensive, limiting their use for novel diagnostic and research applications. Additionally, existing technologies are often limited by short read lengths, which may present problems to certain quantitative sequencing applications. One such application is Differential Expression Analysis, in which RNA-Seq is performed in paired samples under different experimental conditions to identify differences in gene expression. In this study, an Oxford Nanopore Technologies sequencing platform was used to conduct a differential expression study to identify Notch targets. Notch is a transcription factor that regulates numerous functions related to cellular growth and development, and misregulations in the Notch pathway can lead to developmental disorders and cancer. Nanopore Sequencing offers a cheaper and potentially more effective way to conduct research on Notch-mediated expression. It was found that while nanopore sequencing offers a cheaper alternative to existing methods, additional development of the technology is required to perform at the same level as current research standard platforms in differential expression

Rapid on-site detection of harmful algal blooms: real-time cyanobacteria identification using Oxford Nanopore sequencing

With the increasing occurrence and severity of cyanobacterial harmful algal blooms (cHAB) at the global scale, there is an urgent need for rapid, accurate, accessible, and cost-effective detection tools. Here, we detail the RosHAB workflow, an innovative, in-the-field applicable genomics approach for real-time, early detection of cHAB outbreaks. We present how the proposed workflow offers consistent taxonomic identification of water samples in comparison to traditional microscopic analyses in a few hours and discuss how the generated data can be used to deepen our understanding on cyanobacteria ecology and forecast HABs events. In parallel, processed water samples will be used to iteratively build the International cyanobacterial toxin database (ICYATOX; http://icyatox.ibis.ulaval.ca) containing the analysis of novel cyanobacterial genomes, including phenomics and genomics metadata. Ultimately, RosHAB will (1) improve the accuracy of on-site rapid diagnostics, (2) standardize genomic procedures in the field, (3) facilitate these genomics procedures for non-scientific personnel, and (4) identify prognostic markers for evidence-based decisions in HABs surveillance

Advances in genomics for adapting crops to climate change

AbstractClimate change is a major threat to food security in a world of rising crop demand. Although increases in crop production have previously been achieved through the use of fertilisers and chemicals for better control of weeds and pests, these methods rely on finite resources and are often unsustainable. Recent advances in genomics are laying the foundations for sustainable intensification of agriculture and heightened resilience of crops to climate change. The number of available high-quality reference genomes has been constantly growing due to the widespread application of genome sequencing technology. Advances in population-level genotyping have further contributed to a more comprehensive understanding of genomic variation. These increasing volumes of genomic data facilitate the move towards plant pangenomics, providing deeper insights into the diversity available for crop improvement and breeding of new cultivars. Genomics-assisted breeding is benefiting from these advances, allowing rapid identification of genes implicated in climate related agronomic traits, for breeding of crops adapted to a changing climate

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R1

Review of state-of-the-art algorithms for genomics data analysis pipelines

[EN]The advent of big data and advanced genomic sequencing technologies has presented challenges in terms of data processing for clinical use. The complexity of detecting and interpreting genetic variants, coupled with the vast array of tools and algorithms and the heavy computational workload, has made the development of comprehensive genomic analysis platforms crucial to enabling clinicians to quickly provide patients with genetic results. This chapter reviews and describes the pipeline for analyzing massive genomic data using both short-read and long-read technologies, discussing the current state of the main tools used at each stage and the role of artificial intelligence in their development. It also introduces DeepNGS (deepngs.eu), an end-to-end genomic analysis web platform, including its key features and applications

Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae

Next-generation sequencing : an eye-opener for the surveillance of antiviral resistance in influenza

Next-generation sequencing (NGS) can enable a more effective response to a wide range of communicable disease threats, such as influenza, which is one of the leading causes of human morbidity and mortality worldwide. After vaccination, antivirals are the second line of defense against influenza. The use of currently available antivirals can lead to antiviral resistance mutations in the entire influenza genome. Therefore, the methods to detect these mutations should be developed and implemented. In this Opinion, we assess how NGS could be implemented to detect drug resistance mutations in clinical influenza virus isolates