On the 27th - 29th November 2018, scientists from the Nanopore Community will meet in San Francisco to discuss their work - register now to be part of the conversation. You can expect plenary and breakout sessions, lightning talks, poster sessions and a conference dinner.
See below to find out what happened at Nanopore Community Meeting 2017 in New York.
- Limited offer: $650 - Sold out
- Poster rate: $674 - Closed
- Standard rate: $749 - 25 tickets left
- Pre-meeting workshop*: $490 - Sold out
* Available to members of the Nanopore Community only. This one-day workshop will take place on 27th November and is an additional cost.
Oxford Nanopore Technologies
Clive is Chief Technology Officer at Oxford Nanopore Technologies. 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 Technologies 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.Workshop agenda Agenda
De novo assembly of an entire Mauritian macaque MHC haplotype with ultra-long reads
University of Wisconsin-Madison
The full ~5Mb macaque MHC genomic region is unexplored, despite its importance in transplantation and immunological research. This region is highly complex, with numerous multicopy genes and copy number variants arising from segmental duplication events. Current macaque reference genomes were constructed from short-read sequencing, leaving them condensed and inaccurate in the MHC. We used Oxford Nanopore Technologies sequencing of high molecular weight gDNA to assemble the full MHC genomic region from a homozygous Mauritian cynomolgus macaque. Sixty Oxford Nanopore runs were performed on a MinION device and resulting reads were mapped to the human hg38 reference genome. Sequences mapping to the MHC region were extracted, and extracted reads were de novo assembled using Canu long-read assembler. The entire genomic MHC region can be resolved into a single assembled contig using ultra-long Oxford Nanopore gDNA sequences alone, and mapping of classical and non-classical MHC genes to the resultant contig is ongoing.
Julie Karl is a Senior Research Specialist at the University of Wisconsin-Madison. Her focus is characterization of the major histocompatibility complex (MHC) in non-human primates, supporting models for HIV, transplantation, and other immunological research. She specializes in developing improved methods for sequencing, assembling, and analyzing MHC genes.Workshop agenda Agenda
Signal analysis using nanopolish
Ontario Institute of Cancer Research
Jared Simpson is a Principal Investigator at the Ontario Institute for Cancer Research and has an appointment as an Assistant Professor in the Department of Computer Science at the University of Toronto. Jared's research group develops algorithms for analyzing large-scale sequencing data.Workshop agenda Agenda
The MinION menagerie: applications of nanopore sequencing for animal health and welfare
The Roslin Institute
Animal health and welfare are major concerns both for livestock and companion animals. The productivity of livestock relies heavily on the condition of the animal, and anything affecting animal health can be expensive for farmers. Similarly, ill health in companion animals can be costly and distressing to pet owners. The health of animals can be negatively impacted by variations in their genomes and through microbial activity. This talk will explore the variety of applications for nanopore sequencing to better understand animal health, and the specific benefits of this technology for these purposes. Applications include diagnosis and anti-microbial profiling of infections in dogs, assembly of whole genomes from rumen microbiome samples in cattle, investigation of a genomic region associated with respiratory distress in dogs, and ultra-long read sequencing for the assembly of a wild suid genome to better understand response to local disease challenges in pigs in Africa.
Amanda Warr recently completed her PhD at The Roslin Institute in Edinburgh, UK. Her PhD research involved using genomics to investigate reproductive traits in pigs and reassembling the pig genome using long-read sequencing. Although this work was primarily in bioinformatics, she also spent time in the lab using the MinION and training others to use the sequencer. She has accumulated a number of MinION-related side projects and collaborations including work in a variety of species on anti-microbial resistance, viral epidemiology, genome assembly in mammals and microbiomes, and diagnostics. Currently she is employed as a Postdoctoral Research Fellow at The Roslin Institute in the groups of Mick Watson and Christine Tait-Burkard with main projects focussing on functional genomics in chickens and tracking the spread of Porcine Reproductive and Respiratory Syndrome Virus in the Philippines.Workshop agenda Agenda
High-throughput targeted nanopore sequencing of single cells
Garvan Institute of Medical Research
High-throughput single-cell RNA sequencing is a powerful technique but only generates short-reads from one end of a cDNA template, providing incomplete transcript coverage and limiting the reconstruction of highly diverse sequences such as antigen receptors expressed by lymphocytes. To overcome this limitation, we combined targeted cDNA capture and long-read Oxford Nanopore sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate, full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7,138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Despite the many benefits of capture sequencing, our PromethION runs and bioinformatics development for efficient high-throughput barcode recovery suggests that full-length transcriptome characterisation of thousands of single cells will soon be achievable with nanopore.
Martin is Head of the Genomic Technologies program at the Kinghorn Centre for Clinical Genomics, located at the Garvan Institute of Medical Research in Sydney, Australia. He has been using nanopore sequencing since 2014, with a heavy focus on transcriptomic applications. Martin is a computational biologist from Canada with a background in genomics, microbiology and immunology.Workshop agenda Agenda
From mixed bags to magic bullets: solving challenging problems in genomics
Oxford Nanopore Technologies Ltd
Eoghan Harrington is a Senior Applications Bioinformatician working out of Oxford Nanopore’s New York office. He brings over a decade's worth of experience in genome sequencing to bear on his role in the Genomic Applications Group, a multi-disciplinary team tasked with finding novel uses for Oxford Nanopore Technologies devices and communicating them to a wide audience. To achieve this goal, Eoghan works closely with internal and external collaborators to identify and develop high-impact applications and publicise the results in posters, presentations and scientific publications. After graduating from Trinity College Dublin with a BA in Human Genetics and an Msc. in High Performance Computing, Eoghan went to EMBL Heidelberg to carry out his doctoral research. While there he used comparative genomes to study alternative splicing, in addition to some of the first shotgun metagenomic datasets. He went on to do postdoctoral research in single-cell microbial genomics at Stanford University. Prior to joining Oxford Nanopore Technologies he worked at two start-ups: a leading personal genomics company and an oncology-focused electronic healthcare record and analytics company.Workshop agenda Agenda
Hybrid HMM/neural network decoding of individual nanopore reads
University of California, Berkeley
Ian Holmes studied physics at the University of Cambridge and completed a PhD in Genetics at the Sanger Institute in the early days of the genome projects. He worked at Los Alamos National Lab and the University of Oxford before his current faculty position at UC Berkeley, held since 2004. His group has developed the JBrowse genome browser and various statistical inference algorithms used in molecular evolution and bioinformatics. He is also a recovering indie game developer.Workshop agenda Agenda
Mosquito Y chromosomes
Zhijian Jake Tu
Only female mosquitoes bite and transmit pathogens that cause many diseases, including malaria and Zika. A dominant male-determining factor (M-factor) from the Y chromosome provides the primary signal that initiates male development in mosquitoes. Such M-factors have been recently discovered in a few mosquito species; however, Y chromosomes are still very difficult to study due to their repeat-rich characteristics and Y chromosomes were entirely missing when the 16 Anopheles mosquito genomes were published in 2015. In this presentation, I will report our recent results towards sequencing the Y chromosomes from divergent mosquito species using Oxford Nanopore technology and other platforms. Various methods, including chromosome quotient and hybrid sequencing followed by trio binning, are used to facilitate Y gene discovery and Y chromosome assembly. Evolutionary insights into the distribution and turnover of Y chromosome genes will be presented and the potential for using Y-linked “maleness” genes to control mosquito-borne infectious diseases will be discussed.
Dr. Tu is a Professor at Virginia Tech in Blacksburg, Virginia. He received a Bachelor’s degree from Peking University and a PhD from the University of Arizona. Currently his research group employs omics and data analytics tools to discover candidate genetic elements that control sex determination and sexual differentiation in mosquitoes. The Tu lab uses biochemical, gene-editing, single-cell and other molecular methods to determine the function of these genetic elements and decipher the mechanism of their action. Because male mosquitoes do not bite, the Tu lab is also interested in translating the fundamental knowledge of mosquito sex determination into novel applications that help control mosquito-borne infectious diseases. Dr. Tu is also a Co-Editor in Chief of Insect Molecular Biology.Workshop agenda Agenda
No assembly required: single nanaopore reads yield complete virus genome sequences from naturally occurring, microbial community DNA
University of Hawaii
Viruses are dynamic elements in ocean ecosystems due to their tremendous numbers, and their roles in promoting cell mortality, lateral gene transfer, and genetic diversification. Although recent metagenomic analyses now are providing new insight into naturally occurring viral diversity, obtaining full length viral genomes from metagenomics short-read assemblies remains challenging, and sometimes uncertain. To circumvent these difficulties, we developed protocols that allowed the acquisition of thousands of full-length virus genomes from naturally occurring phage, derived from single nanopore reads. A combination of careful DNA isolation, along with bioinformatic filtering and binning procedures, generated many different and diverse full-length phage genomes from natural marine populations. In this talk I discuss the approach, methodologies, and preliminary ecogenomic analyses of phage diversity and distribution from the ocean’s surface to 250m deep, using these full phage genome sequences derived from single nanopore reads.
Edward DeLong received his BSc. in Bacteriology at the University of California Davis and completed his Ph.D. in Marine Biology at Scripps Institute of Oceanography at the University of California San Diego. He has worked at the University of California Santa Barbara in the Department of Ecology, the Monterey Bay Aquarium Research Institute, and MIT in the Civil and Environmental Engineering and Biological Engineering. Now a Professor of Oceanography at the University of Hawaii, DeLong serves as co-Director for the Center for Microbial Oceanography: Research and Education (C-MORE) and the Simons Collaboration on Ocean Processes and Ecology (SCOPE). DeLong is an elected Fellow in the American Academy of Microbiology, the American Academy of Arts and Science, the European Molecular Biology Organization, the U. S. National Academy of Science, and the American Association for the Advancement of Science. DeLong’s scientific interests focus primarily on central questions in marine microbial genomics, biogeochemistry, ecology, and evolution. A large part of his efforts have been devoted to the study of microbes and microbial processes in the ocean, combining field-based approaches and genomic technologies. Development and application of genomic, biochemical and metabolic approaches to study marine microbial communities and processes has been a longstanding, central area of interest in his lab. Currently, Delong is coupling high-resolution, oceanographic field surveys with advanced genomic technologies, to map the diversity, variability and activity of marine microbial communities in four dimensions in situ.Workshop agenda Agenda
Transcriptome landscape of the developing olive fruit fly embryo delineated by Oxford Nanopore long-read RNA-Seq
The olive fruit fly (Bactrocera oleae) is the most important pest of cultivated olive trees. We explored transcriptional dynamics in olive fly early embryonic development using a custom cDNA synthesis protocol followed by full-length cDNA-Seq using the MinION which yielded a median of 4.2 million reads across the six timepoints studied. We generated a de novo transcriptome assembly and identified 3553 novel genes and a total of 79,810 transcripts; a four-fold increase in transcriptome diversity compared to the NCBI predicted transcriptome. On a global scale, the first six hours of embryo development were characterized by dramatic transcriptome changes. Clustering of genes based on temporal co-expression followed by gene-set enrichment analysis identified key developmental stage-specific processes. We also evaluated an early access version of the upcoming cDNA PCR sequencing kit (SQK-PCS109) and sequencing using RevD flow cells which showed a simpler workflow and doubled read yields.
Anthony Bayega studied Biomedical sciences at Makerere University in Uganda. He then pursued a Master of Science degree at King’s College London, UK, followed by further postgraduate studies at the National University of Singapore. Anthony is currently a PhD candidate in the Department of Human Genetics at McGill University in Canada under the supervision of Professor Ioannis Ragoussis. His work is focused on using long-read technologies, particularly from Oxford Nanopore, to characterize genomes and transcriptomes of organisms.Workshop agenda Agenda
Beyond gene expression: long-read RNA-sequencing of the cancer transcriptome
Genome Institute of Singapore
The ability of RNA-Seq to generate a high-dimensional, quantitative readout of cells and tissues has made it arguably the most successful functional genomics assay in molecular biology. Currently, the vast majority of RNA-Seq data is generated using short-read sequencing of PCR-amplified cDNA, resulting in systematic biases. A new generation of long-read sequencing technology using nanopores, enables amplification-free, direct sequencing of native RNA. The technology has the potential to overcome the major limitations of short-read sequencing, promising to provide a richer and more accurate readout of the cellular transcriptome. In order to systematically evaluate the nanopore technology, we have generated full-length transcript sequencing data of cancer cell lines using PCR-cDNA sequencing (PCR-cDNA), amplification-free cDNA sequencing (direct cDNA), and direct sequencing of native RNA (direct RNA) on the MinION, GridION, and PromethION platforms. The average read length is above 1,000 bp, with direct cDNA and direct RNA generating the longest reads. A sequencing depth of 1 million reads enables detection of 30,000 distinct transcripts, with replicates showing high levels of consistency between PCR free protocols and sequencing platforms (GridION vs PromethION). Overall, technical biases are significantly reduced compared to short-read sequencing, enabling quantification of transcript and gene level abundance with high accuracy. We demonstrate that long-read sequencing can be applied to patient samples, opening the possibility to study native RNA in a clinical context.
Jonathan is a principal investigator at the Genome Institute of Singapore and the National Cancer Center Singapore. Jonathan's research focuses on computational transcriptomics with a particular interest in genomics technology and translational aspects in cancer. He received his PhD in Computational Biology from the Max Planck Institute for Molecular Genetics in Berlin. Jonathan has studied at Freie Universität Berlin in Germany, Sheffield University in the UK, and Stanford University in USA.Workshop agenda Agenda
Applications of nanopore sequencing for plant pathogen detection
Naktuinbouw (the Netherlands Inspection Service for Horticulture)
The production of pathogen-free plant propagation material and seeds is an essential step towards food security. Classical methods for detection of plant pathogens, such as (q)PCR and ELISA, have a very high accuracy but require pathogen-specific primers, probes and/or antibodies. Direct RNA or cDNA sequencing on the other hand can potentially detect all viable organisms in the sample via their transcripts or RNA-genomes. We are currently evaluating the application of nanopore sequencing for plant diagnostics in order to assist, complement and potentially replace the classical methods. To this end, we are establishing the detection of viral and microbial pathogens by cDNA sequencing of total RNA, sequencing and assembly of bacterial plant pathogens as reference for (q)PCR primer development, as well as using plant transcriptomics in order to identify causal agents of diseases.
Laura Wenzel finished her Master’s degree at Wageningen University earlier this year, during which she specialized in cellular and molecular biotechnology. Her first contact with nanopore sequencing was during her master’s thesis at the Departments of Phytopathology and Bioinformatics in Wageningen, where she studied tomato and pepper endophytes. Since April this year she is a junior researcher in the research and development team at Naktuinbouw, focusing on the applications of nanopore sequencing for plant pathogen detection.Workshop agenda Agenda
Closing highly repetitive bacterial genomes from metagenomic nanopore sequencing
The recovery of complete genomes directly from complex microbial communities in quantity and without isolation has long been a sought-after goal in metagenomic research. We present a novel workflow which achieves this goal using nanopore long-read sequencing. The workflow consists of an optimized high molecular weight DNA extraction approach followed by nanopore long-read sequencing, assembly, and consensus sequence refinement steps. We close several novel bacterial genomes within healthy human gut communities with no need for binning or manual assembly, and compare results to read cloud and short-read approaches. Finished genomes include Prevotella copri, an organism characteristic of non-western gut compositions, with a high incidence of repeated sequences. These repeated elements pose a problem for short-read and read cloud approaches, and have stymied previous metagenomic and isolate assembly efforts. Our method represents an effective, straightforward solution for the complete and efficient de novo characterization of structurally complex bacterial genomes within the gut microbiome.
Ami Bhatt is a physician scientist with a strong interest in microbial genomics and metagenomics. She received her MD and PhD from the University of California, San Francisco. She then carried out her residency and fellowship training at Harvard’s Brigham and Women’s Hospital and Dana-Farber Cancer Institute, and served as Chief Medical Resident from 2010-2011. She joined the faculty of the Departments of Medicine (Divisions of Hematology and Bone marrow transplantation) and Genetics at Stanford University in 2014 after completing a post-doctoral fellowship focused on genomics at the Broad Institute of Harvard and MIT. Prof. Bhatt is a current Damon Runyon Clinical Investigator and has received multiple awards for her academic scholarship, including the Chen Award of Excellence from the Human Genome Organisation (HUGO). Her team’s research program seeks to illuminate the interplay between the microbial environment and host/clinical factors in human diseases. Her translational laboratory develops and applies novel molecular and computational tools to study strain level dynamics of the microbiome, to understand how microbial genomes change over time and predict the functional output of microbiomes. These innovations facilitate much improved measurement of the types and functions of microbes in patients with non-communicable diseases, understanding of the interactions between microbial genes, gene products, and host cells and testing of the impact of microbially targeted interventions in clinical trials. In addition to carrying out research at Stanford University, Prof. Bhatt has active collaborations world-wide including in Nigeria and South Africa. She is committed to ensuring that advances in research touch the lives of individuals in all income settings and so also enjoys volunteering for Global Oncology, a non-profit she co-founded.Workshop agenda Agenda
Nanopore sequencing and rapid fusion testing – a ‘killer app’ in molecular pathology
Brigham and Women's Hospital
Gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions, and for recognizing specific tumor subtypes with significantly varying treatment and prognosis. Assessing for such events in a clinical setting must show high sensitivity and specificity in the face of a specimen that may have low tumor fraction, be quick enough to be used in management decisions, and permit the detection of many possible fusion events. We describe an assay for rapid detection of gene fusions with the Oxford Nanopore MinION sequencing system using anchored multiplex PCR (AMP). We report a high sensitivity and specificity on initial proof of concept testing using clinical specimens. The assay provides an alternative to FISH or cytogenetics testing for smaller labs with low testing volume, and could be more broadly deployed with development of cheaper, smaller scale nanopore sequencing platforms.
William Jeck received his dual degree in medicine and genetics from the University of North Carolina, training in the lab of Dr. Norman Sharpless. He trained in Anatomic Pathology at Massachusetts General Hospital where his research focused on use of nanopore sequencing in molecular diagnostics. He is currently a Gastrointestinal Surgical Pathology Fellow at Brigham and Women’s Hospital.Workshop agenda Agenda
Mitigating pandemic risk with influenza A virus field surveillance at a swine-human interface
Centers for Disease Control & Prevention
Exhibition swine are known to be a source for transmission of zoonotic influenza A virus (IAV) to humans which poses a risk of pandemic. Genomic analyses of IAV swine outbreaks are critical to understanding this risk and are requisite in vaccine generation. We developed and deployed a rapid and portable IAV sequencing pipeline dubbed MIA (Mobile Influenza Analysis). Working overnight at a large swine expo, we collected nasal wipes from 24 pigs and extracted, amplified, and nanopore sequenced the IAV genomes. We completed 13 genomes which included one A(H1N1), one A(H3N2), and an outbreak of 11 A(H1N2) H1 δ 2 lineage IAVs. On-site analysis of the H1N2 δ 2 lineage virus showed a likely mismatch with current vaccine candidates and was emailed to CDC so that it could be used for synthetic candidate vaccine virus development roughly 18 hours after unpacking the lab. Rapid genomic characterization of IAVs, like the data obtained here, provides timely information during an outbreak regarding virus lineage, RNA segment reassortment and signatures of mammalian/human adaptation. Genomic data can also be used to infer antigenic characteristics, identify where/when emerging viruses originated and identify transmission chains, which aid in the control emerging IAV threats.
Dr. Matthew Keller is an ORISE postdoctoral fellow under the mentorship of Dr. John Barnes of the Influenza Genomics Team at the Virology, Surveillance, and Diagnosis Branch of the CDC’s Influenza Division. Dr. Keller received his PhD in Biochemistry and Molecular Biology from the University of Georgia where he studied biofuel production in thermophiles. Dr. Keller joined the CDC in April of 2017 where he is currently investigating nanopore sequencing for the study and surveillance of influenza A viruses. His work entitled “Direct RNA sequencing of the coding complete influenza A virus genome” describes sequencing the first RNA genome in its original form and was recently published in Scientific Reports.Workshop agenda Agenda
Unraveling the mysteries of CBD and THC content with a chromosome resolved Cannabis genome
J. Craig Venter Institute
Human demand for non-psychoactive Cannabis with high concentrations of cannabidiol-acid (CBDA) and low delta-9-tetrahydrocannabinol-acid (THCA) has resulted in admixed populations. The enzymes THCA and CBDA synthase compete for a common precursor and copy number, as well as sequence variation, has been offered as alternative explanations for THCA/CBDA ratio. However, our understanding of the underlying genomic architecture of the CBDA/THCA synthase loci has been limited by the repetitive nature of the Cannabis genome. We assembled a high-CBD strain with Oxford Nanopore reads and resolved the genome into chromosomes with a genetic map, which enabled the resolution of 12 CBDA and THCA synthase cassettes in two linked tandem arrays deep in a low recombination island on chromosome 9. The CBDA/THCA tandem arrays are comprised of 30-80 kb cassettes of specific LTR retrotransposons, suggesting a putative mechanism for copy number variation across cultivars, and shedding light on the storied history of Cannabis breeding.
Dr. Todd Michael is Professor and Director of Informatics at the J. Craig Venter Institute (JCVI) in San Diego, USA. Dr. Michael has led commercial and academic genome centers, and currently directs the JCVI Sequencing Core. In addition, his research group is interested in leveraging sequencing technologies and informatics to understand how information is stored in genomes, such as genome architecture, gene and repeat content, and epigenomic state.Workshop agenda Agenda
Analysis of a synthetic human genome with nanopore sequencing
Altius Institute for Biomedical Sciences
The complexity of human DNA sequences, and occurence of technical errors and artifacts, confounds the analysis of the human genome. However, these errors can be understood and mitigated with the use of reference standards. We have developed internal synthetic RNA and DNA controls, termed sequins (sequencing spike-ins), that can be used to understand and improve nanopore sequencing. Sequins mirror human genomic, transcript and microbial sequences of interest. Due to the chiral properties of DNA, sequins retain the nucleotide content and repetitiveness of the original DNA sequence, and recapitulate many of the same errors and bias during nanopore sequencing. Due to their synthetic sequence, sequins can be added directly to an RNA/DNA sample prior to sequencing, and analyzed as internal qualitative and quantitative controls in the output library. We have built sequins that represent hundreds of clinically-important features of the human genome, including genes and mutations associated with cancer and inherited disease, diverse structural variants, mitochondria and immune receptors. We use these sequins during nanopore sequencing of the human genome where they provide an ideal internal ground-truth set by which we can evaluate the diagnosis of germline and somatic mutations, resolve complex structural variants (such as oncogenic translocations and papilloma viral insertions), and perform rapid HLA typing. We have also developed a set of sequins that comprise a synthetic transcriptome of hundreds of spliced human gene isoforms. When added to RNA samples, these sequins can help measure gene expression, resolve spliced isoforms, and assess the diagnosis of fusion genes in cancer samples. Finally, we have also built sequins that represent a synthetic community of microbial genomes that can assess pathogen detection, resolve strains and variants and enable improved normalization between multiple samples. In each of the above applications, we show how sequins can measure and also mitigate technical errors that occur during nanopore sequencing. By comparison to sequins, we can minimize the impact of base-calling errors, and thereby improve the resolution of difficult and refractory sequences, and ultimately improve diagnostic power and yield. Together, these studies show how reference RNA and DNA standards provide and simple, yet effective approach to improving the standardization, accuracy and performance of nanopore sequencing.
Timothy Mercer is Chief Investigator at the Altius Institute, Laboratory Head at the Garvan Institute of Medical Research and Associate Professor at Faculty of Medicine, University of New South Wales. He has research interests in genome informatics, RNA biology, sequencing technologies and bioinformatics, and has, most recently, pioneered the use of synthetic RNA/DNA standards during the sequencing and analysis of the human genome.Workshop agenda Agenda
High-throughput automation for Oxford Nanopore library preparation
Oxford Nanopore long-read sequencing enables us at Ginkgo Bioworks to create high quality reference genomes for new host organisms. Our ever-increasing number of new partnerships with diverse hosts is driving our demand for Oxford Nanopore sequencing, and library preparation has become a rate-limiting step. We have been developing semi-automated Oxford Nanopore library preparation workflows in order to match our growing demand. Using Oxford Nanopore’s rapid library preparation kits in combination with Ginkgo’s high throughput automation enables us to prepare hundreds of libraries per day. Additionally, through our automatable methods of high molecular weight DNA extraction, we aim to show progress towards an end-to-end fully automated Oxford Nanopore library preparation workflow. It is our hope that in the future, we can apply fully automated techniques across all Oxford Nanopore library preparation workflows in order to increase prepared libraries to thousands per run while decreasing turnaround time.
Rachel Rubinstein is a Test Engineer at Ginkgo Bioworks on the Next-Generation Sequencing team. After receiving her B.Sc. in Biology from Tufts University, Rachel joined the NGS group at Ginkgo where she works on operation and development of high-throughput Illumina DNA sequencing pipelines. She also helps to develop the Oxford Nanopore platform at Ginkgo, using the technology to help onboard and create reference sequences for new host organisms. Additionally, Rachel works to automate Oxford Nanopore protocols in order to be able to bring this technology up to a high-throughput, production-level scale to enable the long-read sequencing of hundreds of different bacterial and fungal microbes at once.Workshop agenda Agenda
Proprotein convertase subtilisin kexin type 9 knockout mice are protected from valvular calcification
Monzino Cardiology Center
Aortic valve calcification (AVC) is one of the most common forms of heart valve disease and affects 3% of the population. No pharmacological treatments have been identified yet; however, proprotein convertase subtisilin kexin type 9 (PCSK9) inhibitors have been proposed to reduce AVC progression. We aimed to evaluate the role of PCSK9 in aortic valve calcification. Total calcium content of aortic valve from Pcsk9-/- mice was significantly lower than WT (p=0.0002). Calcium quantification revealed that Pcsk9-/- VICs were able to calcify at a lesser extent than WT (p<0.01). The implementation of the cDNA-PCR sequencing kit resulted in ~3 Gbases per sequencing run with a coverage of 82%. RNA-Seq of VICs from WT and Pcsk9-/- mice revealed that 363 genes were differentially expressed more than two-fold (adjusted p value<0.05). Functional analysis showed that in Pcsk9-/- VICs upregulated genes coordinate p38 cascade and cytoskeleton modifications, while down-regulated ones control apoptotic signalling pathway, cell proliferation and adhesion, and oxidative stress. These preliminary results show, for the first time, that PCSK9 could play a direct role in AVC and thus, therapies against PCSK9 may be beneficial to patients affected by calcific aortic valve disease.
Dr. Paolo Poggio leads the Aortic, Valvular and Coronary Pathologies unit at the Monzino Cardiology Center since January 2016. The unit undertakes a broad range of basic and translational science research projects in order to improve diagnosis, treatment, and prevention of cardiovascular disease. The main research objectives are to understand the molecular mechanisms involved in the calcification of the aortic valve and discover new biomarkers able to identify patients before any symptoms occur, and study the progression of mitral valve prolapse and investigate new biomarkers able to identify the early phases of the pathology. In addition, the unit also aims to find new biomarkers labelling the ascending aorta condition to aid doctors to decide the correct timing of surgical intervention and recognize new biomarkers able to predict the patient prognosis after coronary artery bypass surgery.Workshop agenda Agenda
Nanopore sequencing of emerging viruses in a “hotspot” – African swine fever and avian influenza in Ukraine
The National Academy of Agrarian Sciences of Ukraine
Emerging viruses often exploit novel ecological niches and infect new host species. Virus epidemics in animals (zoonoses) are highly dependent on interfaces between animal populations to achieve sustained transmission. We have developed nanopore sequencing protocols to study the genetic signatures of two lethal pathogens that recently emerged in Ukraine, from biosafe clinical necropsy samples of total nucleic acids, without virus growth in vivo. For the first time with MinION technology, we sequenced the full 189kbp DNA genome of an African swine fever virus (ASFV) of the virulent Georgia/2007 lineage that has spread throughout eastern Europe, Russia and China. Amplicon panels were developed for on-site genotyping and epidemic tracing, to uncover the mystery of the spread of ASFV in pigs and wild boar. Similarly, multi-segment RNA genomes and defective-interfering RNA species of highly pathogenic H5 AIV from domestic chickens and wild ducks were amplified by RT-PCR, barcoded, and sequenced, revealing origins of avian influenza virus (AIV) in Ukraine.
Dr. Ganna Kovalenko attended the Odessa State Agrarian University in Ukraine and studied veterinary medicine for her PhD, before beginning her research career in the field of parasitology. She is now a researcher in the Laboratory for Animal Disease Diagnostics at the Institute of Veterinary Medicine of the National Academy of Agrarian Sciences of Ukraine (IVM NAAS), where she works on molecular and serosurveillance of zoonotic diseases and sequence analysis of emerging viruses, including African swine fever and highly pathogenic avian influenza. She is a key scientist in an international collaboration with four scientific institutions in Ukraine and the University of Alaska, to build capacity for genotyping emerging pathogens in Ukraine using the MinION.Workshop agenda Agenda
A chromosome-scale assembly of the sorghum genome using nanopore sequencing and optical mapping
Stéphane Deschamps received his Ph.D. in Chemistry and Biochemistry at the University of Oklahoma and is now a Technology Leader in genomics at Corteva Agriscience, Agriculture Division of DowDuPont. His career spans over two decades of experience in the field of genomics and DNA sequencing, starting with his Ph.D. work on the Human Genome Project. His research interests include the development of new methods and applications for next-generation DNA sequencing technologies. He published early studies on the use of Illumina sequencing for SNP discovery and genotyping-by-sequencing in plants, and, later, focused on the development of procedures for targeted genomic libraries and sequencing. More recently, he has been involved in assessing long-read single-molecule sequencing technologies for assembling and characterizing complex plant genomes and transcriptomes.
Workshop agenda Agenda
Multiplex CRISPR-Cas enrichment of clinically relevant genomic repeat structures
With the maturation of long-read sequencing technologies, the application of genome diagnostic tests in patient care are expanding. A promising application is the sequencing of clinically relevant repeat structures in disorders such as Amyloid Lateral Sclerosis (ALS), Huntington's disease, fragile X syndrome, and spinocerebellar ataxias. Increasing number of repeat units within such a repeat locus are indicative of disease severity and age of onset, and therefore of clinical importance. Due to the repetitive nature and length (up to several kilobases) it is hard to determine the exact structure of repeats with traditional sequencing technologies. Long-read nanopore sequencing has the potential to overcome this problem by sequencing the entire disease locus including the full (expanded) repetitive region and associated epigenetic modifications. We present our work on the development and testing of a cost-effective and comprehensive diagnostic test that targets clinically relevant repeat structures in one experiment. The test is based on multiplexed sequence enrichment of repeat loci with CRISPR-Cas technology, according to a protocol developed by Oxford Nanopore Technologies. We used this approach to sequence control samples and patients with expanded repeats, and demonstrate that CRISPR-Cas enrichment combined with nanopore sequencing provides an efficient strategy to sequence clinically relevant genomic repeat structures.
Martin Elferink is a postdoc bioinformatician at the Genome Diagnostics section at the Pharmacy and Biomedical Genetics Division of the University Medical Center Utrecht in the Netherlands. Since 2012 he has worked on the implementation and accreditation of next-generation sequencing into clinical patient care, including gene-panel sequencing, WES, WGS, and NIPT. His current work is focused on innovations in bioinformatics, and the applications of long-read sequencing technologies in genome diagnostics. Martin obtained his PhD at the Animal Breeding and Genomics Centre at the Wageningen University in Netherlands.
Workshop agenda Agenda
Exploring the genomic instability in acute myeloid leukemia by nanopore sequencing
University of Florence
The actual prognostic stratification of acute myeloid leukemia (AML) categorizes patients into favourable, intermediate (IR-AML) and high-risk categories based on conventional karyotyping and additional mutations. Such categorization results in unsatisfying predictions of IR-AML patients’ outcome with a wild-type karyotype at diagnosis, representing one of the main unmet needs in the managing of AML. Furthermore, the 5-20% of patients with myeloproliferative neoplasms, transform to a secondary acute myeloid leukemia (sAML) with an expected aggressive phenotype. Considering that genomic structural variants and copy number alterations are found in a wide range of neoplasms and associated to sAML transformation, the study of the pathologic genome by nanopore technology could help to delineate the landscape of genomic instability in AML, and the following correlation to patients’ outcome could contribute to define a better prognostic stratification.
Niccolò Bartalucci obtained his Biotechnology degree in 2008 and completed a PhD fellowship in Experimental and Clinical Oncology, during which he worked at Institut Gustave Roussy in Paris and University of Florence. He is currently working in the Department of Experimental and Clinical Medicine at the University of Florence, where he is involved in the onco-hematological research field which encompass in-vivo mouse models, cellular and molecular biology.Workshop agenda Agenda
100 genomes in 100 days: The structural variant landscape in tomato genomes
Johns Hopkins University
Structural variations (SVs) are known to be major drivers of quantitative variation in many species. Short-read sequencing has proven valuable for single nucleotide polymorphism (SNP) discovery, but lacks power for more complex SVs. Long-read sequencing has the potential to illuminate this large reservoir of hidden variation, but has previously been too costly and time-intensive to apply at scale. Addressing this critical gap, we have optimized the Oxford Nanopore PromethION to produce read lengths averaging over 30kb and yields up to 100Gbp per flowcell, making it fast and affordable to sequence large numbers of samples. We are using this technology to sequence and understand SVs in 100 varieties of tomato sequenced over about 100 days. Here we discuss 3 important questions: (1) How to select the samples for sequencing that will capture the largest amount of variation; (2) How to rapidly sequence, basecall, and manage the data for 100 genomes; and (3) How to identify SVs across the population using a combination of de novo assembly and read-mapping approaches. Each sample shows ten to forty thousand variants, including many within gene sequences that were not detectable using short-reads. Furthermore, for some variants we have developed CRISPR-based mutants showing novel fruit morphologies and inflorescence branching phenotypes. We believe this approach will become the new gold standard for SV analysis in all species.
Michael Schatz is the Bloomberg Distinguished Associate Professor of Computer Science and Biology at Johns Hopkins University. His research is at the intersection of computer science, biology, and biotechnology, and focuses on the development of novel algorithms and systems for comparative genomics, human genetics, and personalized medicine. In 2015, Schatz received the Alfred P. Sloan Foundation Fellowship to develop computational methods to probe the genetic components of autism and cancer, and in 2014 Schatz received the NSF CAREER award to develop computational methods to study plant and animal genomes using new long-read single molecule DNA sequencing technologies. Schatz joined Johns Hopkins University in 2016, after spending 6 years at Cold Spring Harbor Laboratory, where he remains an Adjunct Associate Professor of Quantitative Biology. Schatz received his Ph.D. and M.S. in Computer Science from the University of Maryland in 2010 and 2008, his B.S. in Computer Science from Carnegie Mellon University in 2000, and spent 5 years at the Institute for Genomic Research (TIGR) in between.Workshop agenda Agenda
From re-tracing history to uncovering architectural secrets: how sequencing is changing Bordetella pertussis research
University of Bath
Long considered highly monomorphic, research into Bordetella pertussis has been revolutionised by sequencing. Progression from allele typing of selected genes to whole genome analysis has revealed an unforeseen level of variation in the bacterium, which is the major cause of whooping cough. Despite a vaccination programme with relatively high global uptake, whooping cough cases have been on the rise since the early 1990s. Large-scale genomic studies can show how the currently circulating population is evolving in response to vaccination, as well as pinpointing the 15th century outbreak when B. pertussis emerged as a global pathogen. In addition, we are now using long- and ultra-longread sequencing to uncover secrets of B. pertussis genomic architecture, including rearrangements and duplication events. In my presentation I will bring together these examples, and more, to illustrate the many ways in which sequencing is changing B. pertussis research and informing our understanding of whooping cough resurgence.
Natalie Ring graduated from the University of Bath with a BSc in Biochemistry in 2012. She then spent four years working at MRC Harwell as a data wrangler for the International Mouse Phenotyping Consortium, as well as completing a post-graduate qualification in Science Communication from the University of Edinburgh. She is currently a PhD student at the University of Bath in the Bagby and Preston groups, studying the genome Bordetella pertussis, the bacteria responsible for whooping cough.Workshop agenda Agenda
How next generation sequencing is shaping the presence and the future of reproductive medicine
University of Oxford
The field of reproductive medicine has undergone significant transformation with the introduction of next generation sequencing (NGS) into several areas of routine clinical practice. Perhaps the most important of these has been the application of NGS to fetal aneuploidy screening, leading to the development of non-invasive prenatal testing (NIPT). This approach involves sequencing of DNA from a maternal blood sample, generating a sufficiently large number of reads that subtle differences in the quantity of reads derived from individual chromosomes caused by fetal aneuploidy can be detected, even when the vast majority of reads are of maternal origin. This technique has enabled early detection of the majority of fetal aneuploid syndromes with high accuracy and without the risks to pregnancy associated with traditional ‘invasive’ prenatal diagnostic methods. The second robust application of NGS involves preimplantation genetic diagnosis (PGD), a technique that requires a biopsy and testing of a single cell from embryos created using in vitro fertilisation (IVF). After removal, the cell(s) are tested for aneuploidy and/or single gene mutations in cases where one/both parents are known carriers. Only those embryos found to be free of aneuploidy/mutation are selected for uteroine transfer, avoiding both pregnancy termination and affected births. Methodologies employed for PGD include: low-pass genome sequencing, counting the number of mapped reads per chromosome in order to detect aneuploidy; targeted sequencing methods to identify inherited mutations in specific genes; deep sequencing of the mitochondrial genome, revealing mtDNA mutations and quantifying heteroplasmy. While PGD has proven to be a valuable reproductive option for couples at high-risk of transmitting a genetic disorder, this approach is often unsuccessful due to the fact that some treatment cycles do not yield any unaffected embryos. The possibility of genome editing, such as CRISPR-Cas9, could in theory circumvent this difficulty, correcting mutations and thereby rescuing embryos that would have been discarded after PGD. This may potentially improve the likelihood of a positive reproductive outcome for patients. With the numerous technical challenges that CRISPR-Cas9-like approaches are currently facing, the applications of NGS remain instrumental to the assessment of its safety, efficacy and off-target consequences prior to any clinical application can be considered.
Nada is a third year PhD student in Obstetrics and Gynaecology at the University of Oxford. In 2015, she completed the MSc in Clinical Embryology, University of Oxford, and since then she has been working clinically with couples undergoing fertility treatments where she analyses their embryos for known genetic abnormalities. Her doctoral research focuses on the development of novel protocols for the detection of aneuploidies and genetic defects in human gametes and IVF embryos, with the intention to provide innovative solutions for pre-implantation genetic diagnosis and screening, particularly with the use of new technologies such as next-generation sequencing. Recently, Nada has been working on questions relating to the DNA repair mechanisms in early preimplantation embryos with the utilisation of germline genome editing (GE), and she is interested to investigate the possibility of using the GE technology to prevent genetic disease in human IVF embryos.Workshop agenda Agenda
How nanopore sequencing is changing psychiatry
University of Oxford
Schizophrenia diagnosis is based on descriptions of symptoms that have changed very little in the past 100 years. Whilst genome-wide association studies have linked genes, including the calcium channel CACNA1C, to the risk of schizophrenia, the underlying molecular mechanisms are not understood. The disease-associated CACNA1C variants are within non-protein coding introns so may instead be regulating gene expression via mRNA splicing. Alternative splicing is known to change the activity of the CACNA1C protein, so characterising full length mRNA molecules is key to understanding gene function. Standard RNA-seq struggles to identify full length mRNA isoforms, but long-range Nanopore sequencing of mRNA has enabled us to characterise the splice isoform profile of CACNA1C. We have identified 38 novel exons and 83 high-confidence novel isoforms in the human brain, greatly increasing the known splicing complexity from the previously predicted 40 isoforms. We are now using nanopore sequencing to investigate differences in CACNA1C splicing between healthy people and patients to understand the cellular mechanisms underlying schizophrenia and identify treatment targets. Nanopore sequencing is transforming psychiatry by taking us beyond disease-associated genes to how these genes are functioning and towards life-changing treatments for schizophrenia.
Nicola Hall is a postdoctoral researcher at the University of Oxford, Department of Psychiatry in the Tunbridge group. She is using her background in molecular biology and RNA sequencing to investigate gene expression in psychiatric disorders, currently focusing on alternative splicing of the calcium channel CACNA1C, implicated in schizophrenia and bipolar disorder.Workshop agenda Agenda
Lineage calling using short k-mers can identify drug resistant clones in minutes
Harvard T. H. Chan School of Public Health
Surveillance of circulating drug resistant bacteria is essential for healthcare providers to deliver effective empiric antibiotic therapy. However, the results of surveillance may not be available on a timescale that is optimal for guiding patient treatment. We present a method for inferring characteristics of an unknown bacterial sample by identifying the presence of sequence variation across the genome that is linked to a phenotype of interest, in this case drug resistance. We demonstrate an implementation of this principle using sequence k-mer content, matched to a database of known genomes. This technique can be applied to data from an Oxford Nanopore device in real time and is capable of identifying the presence of a known resistant strain in five minutes, even from a complex metagenomic sample. This flexible approach has wide application to pathogen surveillance and outbreak response, and may be used to greatly accelerate diagnoses of resistant infections.
Bill Hanage is an evolutionary biologist and epidemiologist who uses genomics and computational biology to study (mostly) bacterial pathogens. His honors include the Fleming Prize from the Microbiology Society.Workshop agenda Agenda
Nanopore goes viral (RNA)
University of Arkansas for Medical Sciences
RNA viruses, particular single-stranded (ss)RNA viruses, cause many emerging and re-emerging infectious diseases. Our current knowledge of the RNA viruses is rather fragmentary. Genome sequences of many viruses are available and can be used for biosurveillance, diagnosis, and antiviral drug discovery. Still, outbreaks of viral diseases are a never-ending challenge. In 2017, Oxford Nanopore technology released direct RNA sequencing technology that possibly reveals additional layers of viral genetic modulation such as RNA base modifications and full-length transcriptomic architecture. Using direct RNA sequencing by MinION, full-length RNA in native form can be delivered in real-time. To obtain more complete genetic information of RNA virus, we developed a simple protocol that can be used to capture several layers of genetic information such as i) detect complex population of RNA viruses, (ii) sequence native RNA form enabling study of RNA modifications and RNA structure forming, iii) detect genome, subgenome/transcript simultaneously through orientation specific sequencing, iv) enabling real-time detection. We applied this for a set of six diverse ssRNA viruses (positive-strand, negative-strand, segmented/non-segmented genomes). All six viruses were passed through nanopores within 2 minutes after sequencing start. We observed several individual RNA reads were longer than 10kb which almost covered the viral genomes. Mapping of the sequences on the six genomes showed 99%-100% recovery. The directional-specific RNA sequences obtained can be used to profile the transcriptome of the viruses directly, especially the transcriptome of the negative-strand RNA viruses. Native RNA data of viral genomes and transcripts viruses are now available for investigation of RNA modifications and RNA structures.
Dr. Thidathip Wongsurawat (Tip) is a post-doctoral researcher in the Department of Biomedical Informatics at the University of Arkansas for Medical Science (UAMS). Her research interests focuses on utilizing the Oxford Nanopore technology to study viruses, the virome and epitranscriptome. Currently, she trains undergraduate and graduate students, researchers and physicians to use nanopore technology to sequence the genome and transcript of virus, bacteria, fungi, plant, mouse, and human cell-lines, as well as the metagenome from different types of clinical samples.
Workshop agenda Agenda
DNA sequencing: changing the game of forensic investigation
University of Texas at Arlington
DNA identification was a game changer for forensic analysis. Due to the implementation of DNA analysis, we are now able to connect suspects to crimes, victims to crimes, and crimes to crimes. We can identify missing persons and unidentified remains using familial associations. As DNA sequencing technologies improve, so do the capabilities of forensic laboratories. Thanks to sequencing, we can identify bio-terroristic materials, such as deadly spores and intrusive plant species, as well as link pollen grains on a vehicle to a remote crime scene. Furthermore, only with sequencing can you unambiguously identify alleles in short tandem repeats that would be indistinguishable using contemporary methods. Continued advancements have already allowed for enrichment free mitochondrial DNA analysis and rapid identification of individuals from a database using single nucleotide polymorphisms, and platforms such as the Oxford Nanopore MinION show promise for even farther advancements, including direct analysis of forensic samples, including blood, semen, and buccal swabs, microbial identification for bioterrorism and geolocation, as well as in-field testing. In addition to the evolution in technology and capabilities in forensic laboratories, the common utility of third party DNA sequencing companies for personal use, such as 23andme and ancestry.com, has created yet another information hub for investigative genetics. Data obtained from these companies is often open-access information, and investigators are taking advantage, using results to locate leads through familial matches. In this talk, I will discuss the evolution of forensic DNA analysis, the current opportunities afforded by DNA sequencing replacing capillary electrophoresis, and the future potential for laboratories and investigators when assessing genetic evidence.
Dr. Roxanne Zascavage is an Assistant Professor in the Department of Criminology and Criminal Justice at The University of Texas at Arlington (UTA), with a core focus on forensic science instruction and research. Prior to joining the team at UTA, she was a postdoctoral associate at the University of North Texas Health Science (UNTHSC), from where she had received her Master of Science Degree in Forensic and Investigative Genetics, and her Ph.D. in Biomedical Sciences with a concentration in Molecular and Medical Genetics. Dr. Zascavage continues to collaborate closely with UNTHSC to evaluate novel DNA sequencing technologies, particularly utilizing Oxford Nanopore technology for forensic application and creating a working platform for small to mid-sized laboratories. She and her colleagues at UNTHSC and Baylor College of Medicine were recently awarded a grant from the National Institute of Justice for Research and Development in Forensic Science for Criminal Justice Purposes. The title of their project, set to begin in January 2019, is ‘DNA typing strategies via real-time nanopore sequencing for forensic analysis’.Workshop agenda Agenda
Clinical sequencing of pathogens and human host responses in acutely infected patients
University of California, San Francisco
Metagenomic next-generation sequencing (mNGS) and RNA gene expression sequencing (RNA-Seq) are emerging approaches for diagnosis and characterization of infectious diseases. Nanopore sequencing on the MinION is an attractive technology for implementation of mNGS and RNA-Seq in clinical settings given the accelerated sample-to-answer turnaround times and capacity for real-time analysis of clinical sequencing data. Acute infectious syndromes such as sepsis, encephalitis, and pneumonia can be caused by a broad number of potential pathogens. Combined with the diversity of the patient host response and rapid evolution of severe illness, these infections are exceptionally challenging for physicians to accurately identify, diagnose, and treat, Here we will describe novel nanopore library preparation protocols for (i) shotgun metagenomic analysis (MSSPE, “metagenomic sequencing with spiked primer enrichment”), (ii) 16S ribosomal RNA (rRNA) microbiome analysis, and (iii) RNA-Seq analysis (single tube reactions) of clinical samples from infected patients, with the goal of pathogen identification and host response characterization for time-critical clinical applications. Coupled to an automated bioinformatics pipeline for real-time sequencing analysis (SURPIrt), we will show that this approach is promising in simultaneously characterizing pathogens and host responses in critically ill patients so that they can be rapidly and accurately identified for targeted treatment.
Charles Chiu, M.D./Ph.D. is 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. He is the principal investigator of the nationwide, multi-site “Precision Diagnosis of Acute Infectious Diseases” study (June 2016 – July 2017) to investigate the clinical impact and cost-effectiveness of a validated metagenomic next-generation sequencing assay for diagnosis of encephalitis and meningitis in hospitalized patients. Chiu currently heads a translational research laboratory focused on next-generation sequencing assay development for infectious disease diagnostics, investigation of emerging pathogens, including Borrelia burgdorferi (Lyme disease), Ebola virus, enterovirus D68, and Zika virus, and clinical/public health applications of new diagnostic technologies such as nanopore sequencing. His work is supported by funding from the National Institutes of Health (NIH), Abbott Laboratories, Department of Defense, NASA/Translational Research Institute, philanthropic grants (Sandler, Bowes, and Schwab Foundations), and the California Initiative to Advance Precision Medicine. Dr. Chiu has authored more than 70 peer-reviewed publications, holds over 15 patents and patent applications, and serves on the scientific advisory board for Mammoth Biosciences and Therabio, Inc.Workshop agenda Agenda
The long short cut to plant biodiversity
Institute of Research for Development (IRD)
The tropics are a hotspot of plant and animal biodiversity. This biodiversity, still largely under described, is highly threatened by global changes like deforestation or global warming. New sequencing approaches can help speed up the identification and discovery of new species. In plants, using full chloroplasts or plastomes have been suggested as the ultimate barcode for plant species discovery. We developed a protocol for in-solution enrichment hybridization capture of long DNA fragments useful to sequence complete chloroplast genomes using the latest available Oxford Nanopore MinION platform. We then applied this protocol to six non-model monocot species in grasses and palms. Our protocol successfully captured long-read chloroplast fragments up to 20 kb. De novo assembly was improved but remains challenging. This new protocol is intended to allow complete recovery of whole chloroplast genomes of closely related species in phylogeography studies using long-read sequences combined with de novo assembly.
Thomas Couvreur is a researcher and botanist at the Institut de Recherche pour le Développement (IRD), based in Montpellier, France. His main interest lies in understanding the evolution, resilience and diversity of tropical biodiversity, and rain forests in particular, one of the most complex and diverse ecosystems on the planet. Thomas undertakes research in taxonomy, molecular phylogenetics and phylogeography using DNA sequence data, morphological evolution, and modelling of species distribution at different time intervals.Workshop agenda Agenda
The world is your MinION
Garvan Institute of Medical Research
Getting started with Oxford Nanopore sequencing has never been easier. However the ambitious goals of sequencing anything, anyone, anywhere, in real-time, carry technical challenges for the users. These include computer infrastructure, data management, bioinformatic tools and pipelines. Our lab has grown from a single MinION device to being the first certified GridION and PromethION service provider in Australia and was the first to break the megabase with ultra-long reads. We are certified in DNA, cDNA and direct RNA and have sequenced bacteria, fungi, viruses, spiders, plants, and a variety of human samples, including single-cell transcriptomes and complex cancer genomes. We have also tackled some difficult bioinformatic challenges and incorporated raw signal data into our analyses. Through this experience, we have learned some valuable lessons, as well as some tips and tricks to be shared that should prove useful for any stage of adoption of this transformative technology.
James Ferguson is a Genomic Systems Analyst in the Genomic Technologies Group at the Kinghorn Centre for Clinical Genomics, located at the Garvan institute of Medical Research in Sydney, Australia. With a background in clinical pathology testing, algorithm development, and infosec, James applies his unorthodox skills developing new bioinformatics tools, as well as designing and supporting sequencing infrastructure.Workshop agenda Agenda
Low-cost rabies virus sequencing around the world using the Oxford Nanopore MinION
Centers for Disease Control & Prevention
For many countries with a high burden of canine rabies, control and eradication efforts are hampered by a lack of information about the distribution and spread of the rabies virus. Rapid, cost-effective sequencing could provide critical information about viral evolution, virus variants and virus-host relationships that is needed to inform control and vaccination strategies. The Oxford Nanopore MinION allows for cost-effective portable sequencing with real-time data analysis. We evaluated the MinION for routine rabies sequencing and portable sequencing. More than 200 divergent rabies virus isolates were sequenced in Guatemala, India, Kenya, Vietnam and the United States. Consensus sequences achieved high accuracy compared to Illumina and Sanger methods. Multiplexing reduced costs to well below other sequencing methods. Phylogenetic analysis provided insight into rabies distribution in regions where rabies sequencing was not possible in the past. Taken together, our evaluation suggests the MinION can produce informative rabies sequences from clinical and field samples.
Dr. Crystal Gigante is an ORISE postdoctoral fellow under the mentorship of Dr. Yu Li and Dr. Suxiang Tong at the Centers for Disease Control and Prevention. Dr. Gigante received her PhD in Cellular, Molecular, and Developmental Biology and Biophysics from Johns Hopkins University, where she studied the role of a nuclear structural protein in developmental gene expression. Dr. Gigante joined the Poxvirus and Rabies Branch at CDC in June of 2016, where she is working to develop molecular-based diagnostic and sequencing techniques for detection and characterization of rabies virus and poxviruses. Dr. Gigante and Dr. Li recently traveled to Guatemala, Kenya, India, and Vietnam to validate a low-cost rabies virus sequence typing method using nanopore sequencing.Workshop agenda Agenda
Application of wavelets to the base-calling of nanopore sequencing data
University of Saskatchewan
Nanopores are providing a disruptive paradigm for sequencing. Opportunities are opening up for anyone to consider sequencing anything, anywhere and being able to work with data in real-time. A major challenge in using nanopores for sequencing is accurately interpreting the electrical signal in order to identify the molecules transiting, or interacting with the nanopore over time. We have been exploring the use of wavelets to process nanopore signal data. Wavelets are of particular interest in signal processing because a wavelet can localize, in time, where the frequency of a signal changes. In the case of nanopore signals, changes in frequency may be coincident with a nucleic acid sequence ratcheting through the nanopore. In this presentation we will demonstrate how wavelets can be used to first de-noise and then to segment nanopore signals. These wavelet approaches are relevant to improving the accuracy of nanopore sequencing as they may provide a cleaned-up signal for training and an alternative approach for segmentation.
Matthew Links is an Assistant Professor in the Department of Animal and Poultry Science and an associate member in Computer Science at the University of Saskatchewan in Canada. His research program is focused on trying to unravel host-microbiome interactions and since establishing his research group in 2016, his team has begun exploring the use and analysis of nanopore sequencing data. Applications have included determination of calf parentage through sequencing of a bull battery and the associated calf crop, and to confirm a known mutation in an epileptic chicken flock the lab has generated an 8x coverage of the epileptic chicken genome. Their research group has also produced a visualization platform for nanopore signal data and a tool for real-time alerting by email or SMS based on detection of user-specified levels of taxa within a sequencing run (MinION Classification & Alerting System – MICAS). The group has also been exploring the analysis of nanopore signal data using wavelets. There are a number of ways wavelets may be useful to reduce the error rate of nanopore data including de-noising and segmentation of signals prior to applying machine learning approaches for base-calling.Workshop agenda Agenda
Introducing DNA sequencing to the next generation while sailing the Bering sea through a storm
University of Alaska Fairbanks
Every year, research cruises take scientists at sea for weeks to months. During these very active periods of sampling, marine biologists interested in genomics have to postpone the collection of results until they return on shore. The portability of Oxford Nanopore devices could drastically modify this workflow and prop up investigations while sampling efforts are still ongoing. This past October, 11 undergraduate students of the STEMseas and UAF BlaST programs explored marine communities using several MinION sequencing stations and a MinIT onboard the research vessel Sikuliaq. During a week of transit in Alaskan seas, the ship sailed for 3 days through a force 8 storm. Despite winds and seasickness, students sampled several depths of the water column and within the next 48 hours were able to analyze the collected metagenomic data. Stratification of ocean physical characteristics and of microbial communities are normally observed with such sampling. However, samples collected during this transit depicted a homogeneous marine community all along the water column. This result could be explained by the mixing effect of the storm and correlated by oceanographic data. The success of this outreach activity demonstrates the pertinence and ease of Oxford Nanopore technology deployment onboard research vessels to give marine biologists access to real-time data collection.
Anne-Lise began her career as a microbiology laboratory technician in France, where she completed a PhD in Microbiology and Biotechnology in 2009. Originally interested in the evolution of metabolic pathways in prokaryotes, she started field investigation when she joined the University of Alaska Fairbanks as a post-doctorate four years ago. Alaska's seas, streams, soils, and organisms now represent her sampling playground. For the past two years, she has directed her research onto the application of Oxford Nanopore technology for the sequencing of her samples, covering metagenomics, full genome, and targeted approaches. Picking an interest in conservation and environmental DNA, she is currently applying her skills to bringing DNA sequencing into the field and more generally, in remote locations.Workshop agenda Agenda
How nanopore sequencing is changing HLA typing for renal transplants in low income countries
University of Birmingham
Currently, patients in low/middle income countries (LMIC) cannot easily access organ transplantation because of the prohibitive costs of HLA typing out-of-country. If cheap and accurate HLA typing was available in country, this would transform the provision of renal transplantation as a cost effective way to avoid dialysis. We aim to establish “a lab in case” which can be taken to LMIC to improve HLA typing of organs. We have secured funding to test an amplicon sequencing approach on 15 kidney donor samples previously typed with standard methods. Long range PCR will be used to provide more complete sequencing of HLA regions and a typing algorithm will be developed. Once suitability of the MinION for HLA typing is validated it is anticipated that this can then be made available to low income countries as an affordable, fast and accurate means of tissue typing in real time.
Tom Nieto is a surgical registrar with a specialist interest in renal transplantation at the Queen Elizabeth Hospital in Birmingham. He is also a clinical research fellow at the University of Birmingham in the UK where he is a final year PhD candidate. His doctoral research focuses on the epigenetics of Barrett’s oesophagus and its progression to adenocarcinoma. His main research interests are translational medicine and development of near-patient technologies.Workshop agenda Agenda
Exploring mitochondrial genetics with nanopore long-read sequencing
Heteroplasmy in mitochondrial DNA (mtDNA) complicates the analyses of mitochondrial genetics. In contrast to nuclear DNA where there are only two states, homozygous or heterzygous, variations at a locus in mtDNA can take on a range of values. This also presents a problem of accurately identifying haplotypes and accounting for them. This data has relevance for clinical interpretation of the significance of pathogenic variants occurring at low frequencies. We use long-reads from Oxford Nanopore to call variants, identify haplotypes and confirm them by orthogonal methods. We describe various challenges in sequencing mtDNA on the MinION, both experimental and analytical, and our proposed solutions. We also show that this data puts strong constraints on the possible rates of recombination in mtDNA.
Ravi Sachidanandam has worked in a variety of areas, ranging from the first map of human single nuceleotide polymorphisms, mRNA splicing, RNAi, to mitochondrial genetics and T-cell receptor (TCR) repertoire diversity. He has developed several bioinformatic tools (Kismeth, for DNA methylation analyses, SpliceRack for mRNA splicing and RNAi Oligo Designer, amongst others) that are widely used in the field. His lab has also developed methods for sequencing that have become standard in their fields (small RNA sequencing protocol, mtDNA and TCR repertoire sequencing). He co-founded Girihlet Inc. along with Anitha Jayaprakash to make these techniques more widely available and develop clinical applications based on them.Workshop agenda Agenda
Real-time leukemia diagnostics with nanopore
The Ohio State University
Acute myeloid leukemia (AML) is the most common leukemia in adults and, unlike almost all other cancers, diagnosis and initiation of treatment often constitutes a medical emergency. The mainstay of treatment for 40 years has been very intensive chemotherapy that destroys leukemia and normal bone marrow alike, leading to many weeks of hospitalization, infection risks, and a highly variable outcome. In 2017 and 2018, the United States Food & Drug Administration (FDA) approved 4 new targeted therapies for AML, 3 of which are effective only in the presence of specific genetic mutations. Despite the need for rapid molecular diagnosis and treatment initiation, current sequencing techniques typically require a week or more, even at major academic medical centers. We are developing nanopore technology and infrastructure to bring real-time, same-day molecular diagnostics to every oncology office.
James S. Blachly, MD, is an Assistant Professor of Internal Medicine in the Division of Hematology and the Department of Biomedical Informatics at The Ohio State University, as well as a Member of the leukemia research program at The OSU Comprehensive Cancer Center. In his clinical practice, he specializes in acute myeloid leukemia, a rapidly-fatal blood cancer. In the laboratory, he is Director of Sequencing R&D for the multi-PI OSU Experimental Hematology Laboratory, and works collaboratively in computational biology across a variety of disciplines.Workshop agenda Agenda
MinION whole genome sequencing of malaria field isolates
Walter & Eliza Hall Institute of Medical Research
Malaria parasite genomics is essential to track transmission dynamics, infection origins and drug resistance to optimise control and elimination strategies. We developed a protocol for whole genome sequencing of Plasmodium field isolates using the MinION portable sequencer. The protocol includes a novel parasite DNA enrichment method and a modified MinION library prep. From 6 μL of DNA we achieved up to 9 Gb data with up to 70% bases mapping to the P. falciparum reference. Asymptomatic infections produced up to 91% genome coverage at a depth of at least 5 reads and genotypes were highly concordant with Illumina data. Higher density clinical infections produced >99% genome coverage at a mean depth of 30X and data was sufficient to call drug resistance variants within 45 minutes. MinION sequencing has the potential to expedite genomic surveillance of malaria parasites and increase genomics capacity in malaria endemic countries.
Alyssa Barry is a Laboratory Head in the Population Health and Immunity Division at the Walter and Eliza Hall Institute of Medical Research (WEHI) and Associate Professor at the University of Melbourne. Alyssa completed PhD studies in human molecular genetics at the Murdoch Children's Research Institute in Australia, followed by Postdoc appointments in bioinformatics at WEHI, malaria genomic epidemiology at the University of Oxford, UK and New York University School of Medicine, USA. Following this, Alyssa returned to Melbourne and established a malaria molecular epidemiology lab at the Burnet Institute. In 2011, she returned to WEHI as a Laboratory Head where she leads a productive research program focused on the genomic diversity of human malaria and other tropical and neglected diseases. Alyssa’s research has revealed key insights into malaria parasite evolution, transmission patterns, drug resistance and vaccines, in addition to host genetics and immunity. This research is providing a better understanding of disease biology and playing a critical role in control and elimination efforts in malaria endemic countries.Workshop agenda Agenda
Lightning talk: Rapid sample-to-answer for fieldable genomic sequencing-based biothreat identification and biosurveillance
U.S Army Research Development & Engineering Command
Lightning talk: Accurate transcriptome-wide identification of m5C RNA modification events at single molecule resolution from direct RNA sequencing of human cell lines
Indiana University - Purdue University
Lightning talk: Full-length alternative isoform analysis of RNA (FLAIR) for nanopore reads
University of California, Santa Cruz
Lightning talk: Ultra-long read sequencing for whole genomic DNA analysis by nanopore devices
The Jackson Laboratory
Lightning talk: Achievement of rapid whole genome coverage of bacterial pathogens at 1 CFU/mL in blood
Day Zero Diagnostics Inc
Lightning talk: Using long-reads to tell one wtf from another wtf
Stowers Institute for Medical Research
Lightning talk: In cold blood: whole genome sequencing psychrophilic Pseudomonas fluorescens subgroup species responsible for fatal blood transfusion reaction
University of Illinois College of Medicine
Lightning talk: Reference-free multiple signal alignment in squigglespace: the necessity but insufficiency of dynamic time warping barycenter averaging (DBA)
University of Calgary
Lightning talk: Exploring the architecture of organoid genomes with PromethION technology
Cold Spring Harbor Laboratory
Lightning talk: Uncovering RNA modifications from single molecule native RNA sequencing using ELIGOS
University of Arkansas for Medical Sciences
Lightning talk: Nanopore MinION sequencing to detect and sequence full genomes of emerging RNA viruses in avian and marine mammal species
University of Alaska at Anchorage
Lightning talk: Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns
University of California, Santa Cruz
Secret cinema: Bulk yeast species level identification and strain level discrimination using ON-rep-seq
University of Copenhagen
Secret cinema: MinION sequencing and de novo assembly of three isolates of the food- and waterborne parasite Giardia duodenalis
Secret cinema: Decoding the Alaskan soil resistome: using nanopore sequencing to understand antibiotic resistance across a permafrost thaw gradient
University of Alaska Fairbanks
Secret cinema: PromethION sequencing of HER2 breast cancers
Rutgers Cancer Institute of New Jersey
Secret cinema: Gene-specific variation in the kinetics of co-transcriptional splicing
Yale School of Medicine
Secret cinema: Using nanopore technology to sequence RNA in its native form
University of Arkansas for Medical Sciences
Secret cinema: Developing an isothermal-amplification-based consensus sequencing method for metagenomics with nanopore long-reads
Okinawa Institute of Science and Technology
Secret cinema: Offline next-generation metagenomics sequence analysis
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