Nanopore Community Meeting 2017
Metropolitan Pavilion, 125 W 18th St, New York City
30th November - 1st December
Pre-meeting workshop 29th November
PCR-based detection is still the most commonly used method for routine identification of viruses of clinical importance, and this approach is often complemented by Sanger sequencing for further genotype determination. Yet, point mutations and recombination events that occur frequently in the genomes of human viruses can potentially lead to false negative results when PCR-based techniques are uniquely used. Here, we developed a set of assays based on Oxford nanopore technologies to obtain whole genomes of enteroviruses (RNA viruses) from clinical samples. We will present our conclusions about direct RNA sequencing vs. indirect sequencing of RNA (via cDNA synthesis and sequencing), in terms of costs, turnaround time, accuracy, and types of foreseen applications in the clinical setting
Alban Ramette received a PhD degree in Natural sciences from the Swiss Federal Institute of Technology (ETHZ) Zurich Switzerland in 2002. His work mostly focused on environmental microbiology and biostatistical analyses of complex datasets (Michigan State University 2002- 2005; Max-Planck Institute Bremen, Germany, 2005-2014). From 2014-2016, he worked on the epidemiology of child asthma and respiratory diseases at the Institute for Social and Preventive Medicine, Bern, Switzerland. In September 2016, he started the Bioinformatics/Biostatistics group at the Institute for Infectious Diseases, University of Bern, Switzerland. Since then, his group has focused on high-throughput sequencing solutions for clinical applications with strong emphasis on products from Oxford Nanopore Technologies. He brings expertise in NGS analysis and statistical analysis of large datasets from clinical and environmental microbiology. Tuesday, 31 October 17 33 Website: http://www.ifik.unibe.ch/
Hospital acquired pneumonia (HAP), respiratory infection developing > 48 h after hospital admission, affects 1.5% (200k) of hospital inpatients per year in the UK and has a 25-50% attributable mortality rate associated with infection. Current culture based diagnosis has sub-optimal specificity and sensitivity and is too slow (>48 hrs) to impact on patient management. Rapid diagnostics are urgently needed to improve the clinical management of HAP, to improve patient outcomes and antimicrobial stewardship. Shotgun metagenomic sequencing has the potential to change the way we diagnose HAP, with improved speed and accuracy compared to current methods. We have developed and optimised a metagenomics pipeline for the diagnosis HAP including human DNA depletion, pathogen DNA extraction, library preparation, MinION sequencing and Epi2Me analysis. We have applied this pipeline to >50 respiratory samples and compared the results to microbiological culture. The turnaround time from sample to pathogen and acquired resistance gene identification is approx. 6 hours, at least 42 hours faster than culture. The overall specificity and sensitivity of the method for pathogen identification compared to culture was >90%. Analysis of resistance data is still underway, however, the mecA gene was detected in all samples positive for MRSA. Real-time metagenomics has the potential to replace culture for the diagnosis of pneumonia and provides the rapid turnaround necessary for precision management of HAP patients
Dr O'Grady gained his BSc, MRes and PhD in microbiology at the National University of Ireland Galway. He remained in Galway for his first post-doc, continuing his research in food microbiology. This was followed by a two year stint in at Beckman Coulter, developing rapid diagnostic tests for infectious diseases. Dr O’Grady then returned to academia, taking up a post-doc position at University College Tuesday, 31 October 17 31 London on TB diagnostics. In January 2013 he was appointed Assistant Professor in Medical Microbiology at the University of East Anglia, UK and was promoted to Associate Prof in August 2016. His research continues to focus on the rapid molecular detection of pathogens, with a particular focus on rapid metagenomics sequencing based infection diagnosis.
The Gastrointestinal Bacteria Reference Unit at Public Health England (PHE) implemented whole genome sequencing (WGS) for outbreak detection and routine surveillance using the HiSeq Illumina platform in 2014. Recently, we explored ways of integrating Oxford Nanopore Minion Technology into our routine service for typing Shiga Toxin producing E. coli (STEC). A pilot study showed that outputs from the Minion platform could be incorporated into the existing PHE bioinformatics pipelines, and integrated with Illumina data. STEC outbreak clusters were successfully identified and investigated using a combination of Illumina and Minion data. Furthermore, analysis of the Minion WGS data facilitated the analysis of the prophage and plasmid content of the STEC isolates, and may provide important clues to the source and transmission of this clinically significant zoonotic, foodborne pathogen.