Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens.

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing Escherichia coli and Listeria monocytogenes by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms.

Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products.

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resistance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven. We present a metagenomics approach capable of delivering the proof of presence of a GMM in a microbial fermentation product, with characterization based on the detection of AMR genes and vectors, species and unnatural associations in the GMM genome. In our proof-of-concept study, this approach was performed on a case with a previously isolated and sequenced GMM, an unresolved case for which no isolate was obtained, and a non-GMM-contaminated sample, all representative for the possible scenarios to occur in routine setting. Both short and long read sequencing were used. This workflow paves the way for a strategy to detect and characterize unknown GMMs by enforcement laboratories.

Patient empowerment for cancer patients through a novel ICT infrastructure.

As a result of recent advances in cancer research and "precision medicine" approaches, i.e. the idea of treating each patient with the right drug at the right time, more and more cancer patients are being cured, or might have to cope with a life with cancer. For many people, cancer survival today means living with a complex and chronic condition. Surviving and living with or beyond cancer requires the long-term management of the disease, leading to a significant need for active rehabilitation of the patients. In this paper, we present a novel methodology employed in the iManageCancer project for cancer patient empowerment in which personal health systems, serious games, psychoemotional monitoring and other novel decision-support tools are combined into an integrated patient empowerment platform. We present in detail the ICT infrastructure developed and our evaluation with the involvement of cancer patients on two sites, a large-scale pilot for adults and a small-scale test for children. The evaluation showed mixed evidences on the improvement of patient empowerment, while ability to cope with cancer, including improvement in mood and resilience to cancer, increased for the participants of the adults' pilot.

TARGETING THE 16S RRNA GENE FOR BACTERIAL IDENTIFICATION IN COMPLEX MIXED SAMPLES: COMPARATIVE EVALUATION OF SECOND (ILLUMINA) AND THIRD (OXFORD NANOPORE TECHNOLOGIES) GENERATION SEQUENCING TECHNOLOGIES.

Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S~rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99\% of reads were correctly classified at the genus level, up to $\approx$40\% were misclassified at the species level. Both~technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.

Nanopore sequencing technology: a new route for the fast detection of unauthorized GMO.

In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region. This DNA walking is performed on transgenic elements, commonly found in GMO, that were earlier detected by real-time PCR (qPCR) screening. Previously, we have demonstrated the ability of this approach to detect unauthorized GMO via the identification of unique transgene flanking regions and the unnatural associations of elements from the transgenic cassette. In the present study, we investigate the feasibility to integrate the described workflow with the MinION Next-Generation-Sequencing (NGS). The MinION sequencing platform can provide long read-lengths and deal with heterogenic DNA libraries, allowing for rapid and efficient delivery of sequences of interest. In addition, the ability of this NGS platform to characterize unauthorized and unknown GMO without any a priori knowledge has been assessed.

Coronary artery treatment with paclitaxel-coated balloon using a BTHC excipient: clinical results of the international real-world DELUX registry.

AIMS: Safety and efficacy of percutaneous coronary interventions using the Pantera Lux paclitaxel-coated balloon have been demonstrated in the PEPPER first-in-man trial. This prospective, multicentre, clinical registry aims to evaluate its safety and efficacy in an international real-world setting in a larger cohort of patients. METHODS AND RESULTS: Between April 2010 and April 2011, 1,064 patients were treated for predominantly diffuse and proliferative in-stent restenosis of bare metal stents (BMS-ISR) and drug-eluting stents (DES-ISR), or for de novo lesions. Clinical device success was obtained in 98.2% of the patients. The study endpoint was major adverse cardiac events (MACE), defined as a composite of all-cause mortality, non-fatal myocardial infarction and clinically driven target vessel revascularisation, and was 8.5% in the overall, 6.0% in the BMS-ISR, 11.5% in the DES-ISR and 7.0% in the de novo population at six months, and 15.1%, 11.6%, 20.6% and 9.4% at 12 months, respectively. Definitive stent thrombosis occurred in 0.4% of the patients within 12 months. CONCLUSIONS: Safety and efficacy of the Pantera Lux paclitaxel-coated balloon was confirmed in a real-world setting with low major adverse cardiac event rates in patients with in-stent restenosis or de novo lesions. (ClinicalTrials.gov: NCT01081366).