ZiBRA project: real-time sequencing of Zika virus in Brazil.
A revolution is occurring in genomic epidemiology. Recently, real-time portable genome sequencing using the Oxford Nanopore MinION device was successfully used to characterize the genetic diversity of the Ebola virus outbreak in Guinea. We are extending this ground-breaking achievement to Zika virus by establishing up the ZiBRA project - a mobile portable genome sequencing laboratory in Brazil. Through collaboration with Instituto Evandro Chagas and Fundação Oswaldo Cruz public health laboratories in Brazil we will perform real-time genomic surveillance of Zika, Chikungunya and dengue viruses, covering a broad geographical region including historical samples, and from patients with a range of clinical presentations. Data will be subject to open release as it is generated with project progress and discussed via the applicants Virological.org forum. This effort will serve as a beacon for open science during a public health emergency.
Nuno’s research focuses on investigating the evolution and molecular epidemiology of rapidly evolving viruses, particularly arboviruses such as Zika, dengue and Chikungunya viruses. He is interested in understanding the factors driving the ignition, establishment and spread of human viruses by integrating genetic, spatial and ecological information. This information could be useful to aid anticipation of virus spread and to design targeted public health responses.
The eXtreme Microbiome Project (XMP) Presents: The Mystery Of The Pink Lake.
The eXtreme Microbiome Project (XMP) is a global scientific collaboration to characterize, discover, and develop new pipelines and protocols for studying novel microorganisms in extreme environments, including “The Door to Hell” gas crater in Turkmenistan, permafrost soils beside frozen mammoths, blood-red waterfalls from Antarctica, and even samples from the International Space Station. Included in this study is Lake Hillier, a bright-pink hypersaline lake located on a remote island off the Western Australian coast. While the pink colour is thought to be caused by salt-loving algae, the actual microbial composition of the lake is unknown. The XMP collected and analysed lake water and sediment samples using a variety of metagenomics techniques, including the Oxford Nanopore MinION. The analysis revealed a surprising range of microbial diversity in the lake, and indicate that many algal, bacterial, and archaeal halophiles contribute to the persistent pigmentation of this pink paradise.
Ken McGrath is the National Sequencing Manager at the Australian Genome Research Facility, based in Brisbane, Australia. He obtained his PhD studying Molecular Pathology in 2005 from the University of Queensland, and has a research background in microbial community genomics, including human and environmental microbiomes and metagenomics analysis. Ken is currently involved with several research projects, including the US-based eXtreme Microbiome Project (XMP), as well as evaluating emerging technologies that can be used to profile the diversity of microbial communities.
Sequencers for soldiers: battlefield genomics.
Why does the military need sequencing, and how will they use it? Recent advances in DNA sequencing technology, such as the Oxford Nanopore MiNION, could transform a previously cumbersome and fixed-location task into a truly mobile prospect. The Defence Science and Technology Laboratory works both to develop its own scientific solutions and to integrate the innovations of the wider community into fieldable technologies for defence and security. My presentation will explore some of the work Dstl is doing to overcome some of the current limitations and how we could apply this work to some example scenarios. I will explore sample collection such as our man-worn aerosol samplers and larger scale environmental collectors. I will discuss our work to develop a simple DNA extraction device, which can be operated whilst wearing bulky personal protective equipment. I will discuss what we need to do to complete a system and how you can help us achieve this.
Claire Lonsdale is a Senior Scientist at the Defence Science and Technology Laboratory. She has a BSc in Microbiology and a PhD in the microbiology of the Human colon. Her work at Dstl has involved extensive research in the areas of molecular biology and sequencing. Current work involves evaluating and developing new techniques and devices for their ability to identify pathogens in environmental samples and diagnostic matrices. Claire’s interests include taking such technologies from the lab to the field and enabling users with little laboratory experience to use them effectively.
Real-time selective sequencing on the MinION
The MinION replaces the conventional model of "sequence followed by analysis to final result" with instant access to data before the completion of a sequencing run. This instant access extends to the analysis of sequence "squiggle" data even before a read has finished traversing the nanopore. We have developed a suite of tools, minoTour, to analyse MinION data in real time and extended them to exploit both ‘Run Until’ and, soon, ‘Read Until’. "Run until" allows the sequencer to switch off after achieving a specific goal, such as depth of coverage. "Read Until" allows individual reads to be rejected from the pore and free that pore to sequence an alternative preferred read. We have developed methods for "Run Until" and "Read Until" in a number of different scenarios including selective small genome sequencing, barcode normalisation and amplicon sequencing. Although read until implementations are limited by compute resource and the ever increasing speed of sequencing, potential applications are increased by enabling the sequencing of regions of DNA from a background pool.
Real-time nano pore sequencing and analysis of plant genomes in a tent in Snowdonia National Park
Rapid CNV characterization of clinical cancer samples on the Oxford Nanopore MinION.
Acute myeloid leukemia is a devastating cancer of the myeloid blood cells where the cells cannot differentiate normally. This disease accounts for ~1% of cancer deaths annually, however the mortality rate exceeds 50% and left untreated can lead to death within a few weeks. Copy number variations (CNV) contribute to cancer pathogenesis, patient prognosis and therapeutic response. Clinical sequencing often takes weeks before actionable results are generated, time which patients may not have. The MinION device could provide clinicians with a tool to generate rapid copy number profiles in a point-of-care setting. Our pipeline generates CNV profiles from cancer samples in three days. Using the widely rearranged SKBR3 cancer cell line as a model we have generated >300k short reads on during a single MinION run followed by CNV analysis by Ginkgo. These profiles identify nearly all CNVs called by Ginkgo from MiSeq libraries. This approach can be used by clinicians to accurately and rapidly characterize CNV profiles from clinical samples and help in informing patient treatment.
Sara Goodwin is the Manager of Technology Development at Cold Spring Harbor Laboratory in New York. In this role she evaluates new and emerging technologies for Next-Generation Sequencing routines and well as develops novel applications and sequencing approaches. Recently her work has focused on long-read sequencing technologies and their applications, in particular long-read sequencing for de novo assembly of large or complex genomes. Prior to her time at Cold Spring Harbor Laboratory, Sara was at The University of Texas, Austin where she focused leveraging sequencing approaches to explore non-specific interactions between cell surfaces and aptamers.
Progress at UC Santa Cruz: Long DNA fragments, tRNA, and Modified Bases
Nanopore strand sequencing is uniquely suited to analysis of long DNA fragments and base modifications. In this presentation, we will discuss recent experiments that demonstrate 99% consensus accuracy for 150kb+ DNA fragments in single MinION runs. We will also discuss single nanopore experiments that detect base modifications on individual native tRNA strands.
Mark Akeson: Beginning in 1996, our group has been developing nanopore devices for analysis of DNA, RNA, and proteins. Our main contributions in this field have been coupling enzyme motors to biological pores to regulate polymer displacement past integral sensors within the pores. More recent experiments have focused on epigenetic modifications of DNA (principally 5-methylcytosine and 5-hydroxymethylcytosine), on tRNA analysis, and on single protein characterization. Over the last nine years we have worked with Oxford Nanopore Technology (ONT) in their efforts to develop a commercial nanopore DNA sequencer.
Nanopore sequencing for metagenomic diagnosis of infectious deseases
Unbiased diagnosis of all pathogens in a single test by metagenomic next-generation sequencing is now feasible, but has been limited to date by concerns regarding sensitivity and sample-to-answer turnaround times. Here we will describe the development, validation, and implementation of a rapid, field-ready assay for differential diagnosis of acute febrile illness on an Oxford Nanopore MinION sequencer that can be performed in under 6 hours. Nanopore sequencing data will be analyzed in real-time on a laptop computer using SURPIrt, a portable version of the SURPI computational pipeline that is currently being implemented for precision medicine diagnosis in hospitals. The eventual goal of these studies is clinical performance validation and deployment at field sites for use in clinical diagnosis and public health surveillance in patients with any unknown febrile illness — including but not limited to infections by Zika virus, Ebola virus, Lassa virus, chikungunya virus, dengue virus, and the malarial parasite Plasmodium falciparum.
Charles Chiu, M.D./Ph.D. is Associate Professor of Laboratory Medicine and Medicine, Division of Infectious Diseases at University of California, San Francisco, Director of the UCSF-Abbott Viral Diagnostics and Discovery Center (VDDC), and Associate Director of the UCSF Clinical Microbiology Laboratory. Dr. Chiu received an MD and PhD in biophysics from UCLA, and trained at UCSF as a resident and clinical fellow in infectious diseases. He currently heads a translational research laboratory focused on clinical diagnostic next-generation sequencing assay development for infectious diseases and investigation of emerging pathogens, including Borrelia burgdorferi, Ebola virus, enterovirus D68, and Zika virus. His work is supported by research grants from the NIH, Bay Area Lyme Disease Foundation, UC Center for Accelerated Innovation, and the California Initiative to Advance Precision Medicine. Dr. Chiu has authored more than 50 peer-reviewed publications, holds over 15 patents and patent applications, and serves on the scientific advisory board for Karius Diagnostics and Rubicon Genomics.
Inside the SkunkWorx
Clive is Chief Technology Officer at Oxford Nanopore. On the Executive team, he is responsible for all of the Company’s product-development activities. Clive leads the specification and design of the Company’s nanopore-based sensing platform, including strand DNA/RNA sequencing and protein-sensing applications with a strong focus on scientific excellence and successful adoption by the scientific community.
Clive joined Oxford Nanopore from the Wellcome Trust Sanger Institute (Cambridge, UK) where he played a key role in the adoption and exploitation of 'next generation' DNA sequencing platforms. This involved helping to set up the world’s largest single installation of Illumina (formerly Solexa) Genome Analyzers in a production sequencing environment, initially used to pioneer the 1000 genomes project.
From early 2003 he was Director of Computational Biology and IT at Solexa Ltd, where he was central to the development and commercialisation of the Genome Analyzer (GA). Solexa was sold to Illumina for $650m in early 2007 after the successful placement and adoption of 12 instruments. The Solexa technology, now commercialised by Illumina, is the market-leading DNA sequencing technology driving the renaissance in DNA-based discovery.
He has a strong background in computer science and genetics/molecular biology and manages interdisciplinary teams including mechanical engineering, electronics, physics, surface chemistry, electrophysiology, software engineering and applications (of the technology). Clive applies modern agile management techniques to the entire product-development lifecycle.
Clive has also held various management and consulting positions at GlaxoWellcome, Oxford Glycosciences and other EU- and US-based organisations. He has worked at the interface between computing and science, ranging from genetics to proteomics. He holds degrees in Genetics and Computational Biology from the University of York.
Genome sequencing a comprehensive communicable diseases solution in the making
Diagnostic and public health microbiology depends on identifying the species, determining the antimicrobial resistance and categorising involvement in an outbreak of infecting pathogens. This is now accessible in one step using whole genome sequencing. There is substantial proof-of-principle work in viruses, bacteria and parasites indicating the advantages of whole genome sequencing of pathogens. The key features required for large scale adoption are achieving greater speed, lower cost and high accuracy of processing clinical samples, if possible, direct from the patient. What is needed is a small, speedier cheaper sequencer requiring nucleic acids prepared by simple quick extraction and fast computationally ‘light-weight’ processing software. The prospects will be highlighted by work on multi-resistant Enterobacteriaceae and TB.
Professor Derrick Crook is currently Professor of Microbiology in the Nuffield Department of Medicine at Oxford University and is a practicing clinical microbiologist and infectious diseases physician at the Oxford University Hospitals NHS Trust. He studied Medicine at the University of Witwatersrand, Johannesburg; obtained the Diploma of Tropical Medicine (London), specialised in internal medicine at the University of Virginia, USA, and completed a fellowship in infectious diseases at the Tufts New England Medical Center, Boston, USA. He obtained his boards in both internal medicine and infectious diseases. He trained in clinical microbiology at the John Radcliffe Hospital Oxford and obtained both his MRCP and MRCPath. He is co-director of the Oxford Biomedical Research, Infection Theme, and leads a large research consortium, Modernising Medical Microbiology, which focuses on translating whole pathogen sequencing into routine practice. The work aims to transform the practice of clinical microbiology, infectious diseases and communicable diseases through implementation of genome sequencing into Public Health England and the NHS.
Applications of Nanopore Sequencing for Infectious Disease Detection.
Though culture methods are the gold standard for detecting clinical pathogenic microorganisms, followed by testing with PCR and microarrays of known pathogens, they are still very limiting. Culture is time consuming, confined by culturability of microorganisms, and gives very narrow information about the sample. We are applying the MinION for shotgun metagenomic sequencing, shortening the detection time and yielding information about substrains and therapeutic resistance from clinical infections. We used nanopore sequencing to detect infectious microorganisms in 3 separate cases: 1) detecting vancomycin resistance in remnant rectal swab cultures, 2) analyzing a clinical timeseries of multidrug resistant hypermucovirulent K. pneumonia to understand antibiotic resistance and containment, and 3) measuring substrain variations of cultured and clinical isolates of influenza strains for real-time surveillance and monitoring of transmission and evolution.
Stephanie Hao is a MSE student in Biomedical Engineering at Johns Hopkins University. She works in Dr. Winston Timp’s lab, where her research interests are focused on applications of Next Generation Sequencing technologies for use in disease detection.
A novel approach to elucidate the genomic structure of plants and pathogens.
KeyGene operates at the forefront with respect to new technologies and innovations and participates as such in the MinION™ and PromethION™ Early Access Programs. Genome sequencing initiatives of large complex genomes typically yield considerably fragmented genome assemblies. Using the Oxford Nanopore sequencing technology that offers ultra- long reads KeyGene aims to produce contiguous, high-quality genome assemblies of plant pathogens and complex plant genomes. In order to obtain high quality libraries for sequencing the DNA integrity and quality are crucial. KeyGene has developed specific knowledge in this area. In 2016 KeyGene started to sequence and de novo assemble the plant pathogen Rhizoctonia solani with a genome size of ~85 Mb. Using the PromethION the next step will be to sequence and assemble the genome of a melon variety (~450 Mb). Progress and challenges in these projects will be presented and discussed during the breakout session.
Alexander Wittenberg graduated as MSc in plant breeding and crop protection at the Wageningen University and subsequently was appointed as PhD student at Plant Research International/Laboratory of Plant Breeding. Here he focused on the development of innovative genotyping methods to study the origin of genome plasticity in crop plants and their wild relatives. In 2007 he joined KeyGene as a researcher were he continued his work on the development and application of molecular marker methods. Alexander developed himself as an expert in the field of Next Generation Sequencing technologies with expertise on a wide range of platforms (Illumina, Pacific Biosciences, Oxford Nanopore Technologies etc.) and applications. Currently Alexander contributes to the development of sequence-based technologies to accelerate the molecular breeding of vegetable and field crops. As a MAP leader he is involved in the evaluation of the Oxford Nanopore sequencing technology in the context of the MinION and PromethION Early Access Programs. Furthermore for two days in the week he is active as business developer genomics and genotyping.