A third generation long-read sequencing approach for the analysis of genomic duplication variants at nucleotide resolution using Cas9 target enrichment
With the widespread use of low-cost genome-wide diagnostic screening tests, unanticipated but possibly pathogenic dosage changes affecting single genes are discovered with increasing frequency. Clinical management demands facile validation of such incidental findings, often necessitating the design of custom variant-specific assays. Although deletion variants can be readily confirmed using a range of next-generation sequencing strategies, characterising duplication variants, at nucleotide resolution, remains demanding. We have addressed this challenge by deploying a novel Cas9 enrichment strategy combined with long-read sequencing with the Oxford Nanopore MinION. We used bulk genomic DNA without the need for PCR amplification. We present the diagnostic resolution of two problematic cases in which incompletely characterised duplication variants had been identified by array CGH. The first patient presented with learning difficulties and autism spectrum disorder but had been found to have an incidental 1.7-kb imbalance which included a partial duplication of VHL exon 3. This was inherited from the patient’s father, who had renal cancer aged 38 years. In the second case, we identified an incidental 200-kb duplication which included DMD exons 30-44. Parental testing was consistent with this variant having arisen de novo. In both cases, the single-molecule sequencing yielded sufficient information to define precisely the architecture of the rearranged region, enabling Sanger sequencing assays across the integration sites and surrounding homologous regions, that likely gave rise to the duplicated sequences. Adoption of this approach by diagnostic laboratories promises to enable rapid and cost-effective characterisation of challenging duplication-containing alleles.
Christopher studied molecular biology and human genetics at the University of Manchester and Mayo Clinic in Florida, USA. He subsequently moved to Leeds to undertake clinical scientist training and attained HCPC registration in 2013. As the NHS lead in the Translational Genomics Unit, he has overseen the clinical implementation of numerous short-read sequencing instruments and next-generation sequencing assays. He is currently a visiting research fellow at the University of Leeds where he is focussed on understanding the clinical utility of long-read sequencing, particularly for the diagnosis of rare Mendelian disease.
An international collaborative effort for infectious disease analyses using MinION
GRAID is the Global Research Alliance for Infectious Disease, a collaborative international effort for infectious disease research using MinION. In this framework, we try to educate researchers and develop the methods and guidelines for field analysis of many aspects of infectious disease. We have conducted four summer schools in three developing countries: Thailand, Indonesia, and Kenya, as part of our efforts to introduce MinION in those countries. We are collaborating with researchers and have produced papers about serotype identification of dengue virus and comprehensive drug resistance identification of malaria parasites. Using MinION and isothermal amplification, we identified serotype of dengue virus in Manado, Indonesia and Hanoi, Vietnam. We found that this method simplifies the amplification of the virus nucleic acid by using only blood or serum and a water bath or a thermal block prior to library preparation. We analyzed 141 Indonesian and 80 Vietnamese patients. The overall successful detection rate was 79% and it depends largely on the viral titer. We also determined that the serotype of dengue virus is different in Indonesia and Vietnam, which is DENV1 and DENV3, respectively. Our next collaborative project is to comprehensively describe the drug resistance of malaria parasites in Indonesia, Vietnam, and Thailand. Here, we used PCR to amplify nine genes correlated to the drug resistance phenotype. We sequenced 118, 11, and 5 samples from Indonesia, Thailand, and Vietnam in multiplex manner and described the drug resistance pattern in each country. We found a position in K13 gene non-propeller region mutated quite frequently from our Indonesian samples. Although we believe that the mutation is not related to artemisinin resistance, we think that the parasites may be on selective pressure due to the artemisinin administration in the region. We also are working with bioinformaticians to develop a graphical user interface tools for researchers or clinicians who are unfamiliar with bioinformatics analysis. We have published Nano Pipe, which serves as an easy to use MinION data analysis tool for a ‘regular’ user. We have ongoing and prospective projects in the framework, such as HLA typing in the severe dengue patients, identification of unknown fever-causing pathogens, and determining the drug resistance pattern in HIV. We are confident that our consortium will make an impact in the infectious disease community to switch to sequencing in the research context while laying some foundations for preventive or therapeutic medicine in the future.
Application of nanopore sequencing in clinical haematology
Professor Schuh completed academic and clinical haematology training in Oxford and in 2006, she was appointed clinical lead for haematology laboratories, including molecular diagnostics, and has also been the clinical lead for chronic lymphocytic leukaemia and other lymphoproliferative disorders for the NHS Thames Valley Cancer Network. Over the past twelve years she has led over 30 early and late phase clinical trials in leukaemia as a principle or national chief investigator. A number of these led to NICE approvals and have changed clinical practice for patients in the UK and worldwide. As a result, she was recently appointed as the Chair of Chronic Lymphocytic Leukaemia Research in the UK by the National Cancer Research Institute. In addition to other national and international roles, she has also chaired the UK CLL Forum since 2016 that promotes training and education, and she has led the UK's guidelines writing group for CLL Therapy on behalf of the British Society of Haematology. Her second research interest is with the development, evaluation and implementation of new technologies for Precision Diagnostics, especially genomics. Her group published the first ever longitudinal study of the changes in the genomic landscape of patients undergoing treatment for leukaemia. She is the lead for the Genomics England Clinical Interpretation Partnership for haematological malignancies. Professor Schuh has received grants from the NIHR, Wellcome Trust, Technology Strategy Board, Cancer Research UK and Bloodwise and she has authored or co-authored over 80 peer-reviewed publications in the last five years.
Comparison of single nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga Toxin producing E.coli
David graduated from the University of Bedfordshire with a BSc in Biomedical Science in 2014 before completing a MSc in Biomedical Science, specialising in Medical Microbiology, from Ulster University in 2015. He then joined the Gastrointestinal Bacteria Reference Unit (GBRU) at Public Health England in London, for the laboratory typing of gastrointestinal pathogens, before moving to the bioinformatics team where he performed data analysis on whole genome sequencing data. David is currently working as a bioinformatician at Public Health England and is a part-time PhD student with the University of Edinburgh, Roslin Institute studying the use of Oxford Nanopore sequencing technologies for the investigation of outbreaks of Shiga-toxin producing Escherichia coli in humans.
CSIR - National Chemical Laboratory
Department of Primary Industries and Regional Development
Direct sequencing of nascent RNA exposes splicing kinetics and order
Human genes contain many long introns with degenerate sequence information at splice sites, requiring sophisticated mechanisms to locate and coordinate the excision of multiple introns within the same pre-mRNA transcript. Fundamental aspects of this process remain unexplored due to a lack of quantitative approaches that monitor RNA processing as transcripts are produced. Here we performed nanopore sequencing of nascent, or newly synthesized, RNA to directly probe the timing and patterns of mRNA splicing. Direct RNA sequencing by the Oxford Nanopore Technologies MinION reveals the native context of long RNA molecules from 3’ to 5’ without amplification-associated biases. By combining direct RNA nanopore sequencing with stringent purification of nascent RNAs, we measure both the active transcription site (nascent RNA 3’ ends) and the splice isoform of single RNA molecules as they are transcribed. Application to human K562 cells reveals that co-transcriptional splicing occurs after RNA Polymerase II has transcribed several kilobases past the 3’ splice site of most introns. We also observe that the order of intron removal is not influenced by transcription direction in human cells. By contrast, we analyzed nascent RNA from Drosophila S2 cells, which have a different gene structure, and found that co-transcriptional splicing occurs more rapidly and in the order of transcription. Treating cells with the splicing inhibitor Pladienolide B abolishes co-transcriptional splicing in both species. Altogether, directly sequencing nascent RNAs through nanopores exposes critical molecular processes that occur during transcription in living cells.
Heather received her bachelor’s degree in Molecular Biology from Princeton University where she worked in Laura Landweber’s lab studying ciliate genome rearrangement. She then spent a year in Bob Langer’s lab at MIT developing a device to predict drug sensitivity in solid tumors. She is currently completing her PhD in Genetics at Harvard University in the lab of Stirling Churchman, working with nascent RNA and nanopore sequencing. She is interested in all aspects of RNA biology and tools to measure co-transcriptional processing.
Dissecting RNA biology, one molecule at a time
The human transcriptome is highly diverse and complex, as evidenced by cell-type specific expression of unique transcript isoforms. In particular, transcripts derived from non-coding regions are the most qualitatively diverse class of genetic elements, encompassing over 70% of the genome. In contrast, about 80% of GWAS SNPs reside in non-coding regions, which suggests long non-coding RNAs may be the missing link, at least to some degree. I will present our recent work on high-resolution cDNA sequencing of non-coding regions associated with neuropsychiatric disorders using targeted sequencing on the PromethION. I will detail the discovery of new non-coding RNAs, mRNA isoforms, long-range exon dependencies, and how these relate to mental health and neurodegeneration. Given the apparent involvement of RNA modifications in neurological diseases, I will then segue into direct RNA sequencing and describe our in vitro strategy to train RNA base callers and detect modified RNA bases. This will include data from our recent preprint on detecting m6A in RNA with 90% accuracy. Finally, I will present how we maximise flow cell output by complementing an innovative RNA barcoding strategy and 'AI'.
Martin Smith 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.
Field-forward sequencing with Oxford Nanopore Technology – a strategy to establish the upside-down mangrove jellyfish Cassiopea xamachana as a bioindicator
The combined relative ease of operation, high throughput and reduced cost of NGS platforms has enabled the coupling of traditional field collection methods with laboratory-based metagenomic approaches to provide a molecular snapshot of species-diversity in a plethora of aquatic environments. However, the often-extensive time-lag between field sampling, sequencing and endpoint bioinformatics analyses precludes the ability to provide a contemporaneous characterization of the target ecosystem, against the backdrop of briskly shifting global climate. Furthermore, the growing decline of healthy aquatic ecosystems due to chemical, physical and biological threats, along with concerns related to invasive species and natural disasters, highlights the critical need for field-forward sequencing protocols to provide rapid characterization of a diversity of environmental systems. The recent publication of the reference genome of the upside-down mangrove jellyfish, Cassiopea xamachana, has gained this emerging model species attention as an indicator species with promising applications for coastal ecosystem management and conservation. Taking advantage of the versatility offered by Oxford Nanopore sequencing, we developed a field-forward DNA environmental metabarcoding strategy to characterize Florida Keys mangrove ecosystems, inhabited by C. xamachana, in the year following the catastrophic landfall of Hurricane Irma in 2017. The prototype for this portable system boasts a low-complexity protocol requiring minimal training for operation, a relatively short sample-to-answer timeframe i.e. several hours, field-forward DNA metabarcoding capabilities in austere environments, manual and/or battery-powered equipment with ease of portability and minimal footprint, as well as multiplexing capabilities for the simultaneous assessment of multiple collection sites and/or genetic markers. We present here the first ever eDNA assessment of C. xamachana populations in several Florida Key coastal environments in the wake of a devastating natural disaster, based on the findings of our inaugural field-forward sequencing study.
Cheryl L. Ames is a National Academies of Sciences (NRC) Postdoctoral Fellow at the United States Naval Research Laboratory and a Research Associate at the National Museum of Natural History, Smithsonian Institution in Washington, DC. As an expert in jellyfish and their evolutionary novelties, such as their venom, vision and sex, Cheryl uses a MinION-based field-forward environmental DNA sequencing protocol to assess biodiversity in marine ecosystems, such as coastal mangroves, the Gulf Stream and public aquaria. Cheryl has a Ph.D. in Biological Sciences from the University of Maryland, USA, a Master’s degree in Marine Biology from the University of the Ryukyus in Okinawa, Japan and undergraduate degrees from Queen’s University and the University of Ottawa in Canada.
Finding disease-causing complex mutations
Martin has a cross-appointment as a researcher at the AIST Artificial Intelligence Research Center, and as a Professor at the University of Tokyo Department of Computational Biology and Medical Sciences. He is broadly interested in analyzing genetic sequences to understand the information encoded in them, their evolutionary history, and their role in disease. He studied Physics and Philosophy at University of Oxford, Mathematics at University of Cambridge, taught English in Beijing, completed a PhD in Bioinformatics at Boston University, and carried out postdoctoral research at the University of Queensland in Australia and RIKEN in Japan.
From amplicons to metagenomes: Long read sequencing the environment
Complex environmental matrices, such as soil, sediment and excreta, are often synonymous with diverse microbial communities. Long read sequencing of DNA extracted from such communities can yield highly contiguous genomic data and provide information on both genetic composition and structure. However, DNA extracted from such matrices is often impure, fragmented and can potentially lack complete representation. Therefore, techniques such as isolation, enrichment and metagenomic assembly are used to answer questions on function and diversity. Here we present the sequencing and assembly of two environmental AMR harboring plasmids and one novel gc-rich genome isolated from the environment. Furthermore, we describe our exploration into the sequencing and analysis of long-range amplicon-based enrichment for AMR associated mobile genetic elements, and undertake metagenomic analysis of the community composition of two fractions of industrial anaerobic digesters. This has permitted us to investigate the evolution and selective drivers of AMR in the environment.
Dr Rob James is currently working as a post-doctoral research fellow with Prof. E. Wellington as lead investigator on the BBSRC funded project; “Mycobacterium bovis and the farmland ecosystem: understanding transmission dynamics between animals and the environment.” This collaborative project between the University of Warwick, the Zoological society of London and Imperial College, aims to identify the environmental reservoirs of infection in agricultural land use types, and routes of transmission between mammalian hosts and the environment. Furthermore, Rob has an interest in the evolution and selection of antimicrobial resistance genes in the environment and has recently undertaken work to quantify AMR gene abundances in farmland and residential areas of Karatchi and Islamabad.
From ancient tomb to animal viruses: mobile suitcase lab for nanopore sequencing at field setting
Nanopore sequencing technology can be applied to identify the pathogen responsible for an outbreak through sequencing all nucleic acids existing in the collected sample in a single run. In addition, it gives insight about the origin and variant of the causative agent. We have established a novel sequencing protocol relying on nanopore sequencing and offline BLAST search beside a microbiome screening of an ancient tomb. The whole procedure was conducted in a solar powered mobile suitcase laboratory, which is easy to use at the point of need. The procedure was completed in 5 hours including extraction, barcoding, sequencing and data analysis, which did not require a bioinformatician. Our protocol enables rapid and reliable foot and mouth disease virus serotyping and the differentiation of the Capri poxviruses (Sheep poxvirus, Goat poxvirus and Lumpy Skin Disease virus). The microbiome composition of the ancient tomb revealed potential threat of respiratory illness due to bacteria from family of Bacillaceae. Furthermore, bacteria from family of Pseudomonadaceae gave hints to the former use of the tomb as a byre.
Dr Abd El Wahed studied veterinary medicine at Mansoura University in Egypt, and received his PhD in biology from Göttingen University, Germany in 2011. He has participated in the development of 30 point-of-care assays for the detection of infectious agents, and In 2013, he was awarded the Young Investigator award from the ASTMH on the establishment of a mobile laboratory for rapid detection of haemorrhagic fever viruses at low resource settings. Recently, he established a mobile suitcase laboratory for rapid detection of viruses, bacteria and parasites. The mobile setup was in field trials in Guinea, Sri Lanka, Nepal, Senegal, Egypt, Bangladesh and Brazil.
Generating high-quality reference human genomes using PromethION nanopore sequencing
In order to catalogue and associate all forms of human genetic variation to health and disease, a new generation of genome sequencing and assembly technologies is required. Current efforts for producing high-quality human genome assemblies of a small number of individuals have focused on costly high-coverage, long-read sequencing and assembly protocols. While this work has been successful in moving toward higher quality reference standards, the overall cost and production time prohibit scaling to hundreds of individuals. New technologies and assembly methods may overcome these barriers through substantial improvements in cost, time and quality. Here we evaluate the PromethION sequencing platform from Oxford Nanopore Technologies to produce reference quality genome assemblies for the offspring of ten parent-offspring trios at a projected cost of around $5K per genome. We are testing the platform’s ability to produce long read, high-quality, and high-coverage genomes with a less than one-week total turnaround time from sample extraction to complete assembly. We evaluate the performance based on assembly accuracy, throughput/timing, and cost; goals that must be met to improve the scale of high-quality genome assembly. Recognizing that even 100kb reads are insufficient to scaffoldmthrough the most repetitive regions of the human genome, we augment this sequencing with a Hi-C long-range library to facilitate scaffolding and haplotype phasing. We aim to produce near-gapless reference genomes at chromosome-level.
Miten is an Assistant Research Scientist at the University of California, Santa Cruz. His research interests include developing methods for long-read sequencing of DNA and RNA, methods for detection of base modification, and software for analysis of MinION data.
Going full circle: Assembly of high-quality, single-contig microbial genomes from the rumen microbiome using long-read sequencing
Ruminants such as cows and sheep are important livestock species. They convert low nutritional value plant matter into high-quality meat and dairy products. Within a specialised stomach called the rumen, microbes ferment the plant matter producing short-chain fatty acids from difficult to digest plant matter. The composition of the rumen microbial community can affect the animal’s health, feed efficiency and level of methane production. Species in the rumen are typically difficult to culture and despite its importance, it remains an underexplored environment. DNA sequencing of the contents of the rumen offers the potential to identify microbial species without culture techniques. Here we sequence cow rumen fluid using Oxford Nanopore sequencing. We show that despite these data coming from a highly complex microbial sample we can assemble high-quality, single-contig whole genomes and plasmids of known and novel species, including numerous circular contigs. Additionally, we compare and validate the assemblies of these genomes with binned genomes generated from short read Illumina assemblies. We show that the long-read assembly out performs the short-read assembly in contiguity and in incorporation of important features such as AMR genes and marker genes..
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 with Mick Watson and Christine Tait-Burkard, with main projects focussing on the rumen microbiome, functional genomics in chickens and tracking the spread of porcine reproductive and respiratory syndrome virus in the Philippines.
Identification of new somatic structural variants and cancer driver genes using long-read nanopore sequencing
Third generation DNA sequencing technologies have been transforming genome medicine and cancer research, producing evidences for structural variations (SV’s) being the common and major driver of complex diseases and tumorigenesis. By taking advantage of the un-parallelled power of long-read and high-throughput capability of the Oxford Nanopore PromethION platform, we investigated the role of SV’s in cancer development. We sequenced DNA obtained from colorectal cancer biopsy and corresponding normal tissue-samples of Han Chinese. Using a comprehensive SV-calling pipeline that consists of ngmlr-sniffle, dynamic filtering, database search and comparison, manual curation, and break point mapping, we obtained high quality SV call sets. By using PCA, population structure, and frequency spectrum analyses, we identified a set of SV’s that are tumor specific. In addition to somatic point mutations in mismatch repair genes that are well known for causing colorectal cancers, we observed complex somatic SV’s that show evidence of chromothriptic rearrangements, the hallmark of the late stage tumors, that were focally localized to a terminal region of a chromosome in colorectal cancer samples. One of the complex somatic rearrangements was linked to the amplification of the gene that is essential for DNA recombination. Furthermore, we also observed a direct link between the expansion of microsatellites and SV’s, suggesting the microsatellite instability might drive the formation of SV’s and cause genome instability in colorectal cancers. Collectively, our results present the power of the Oxford Nanopore PromethION platform for high resolution analysis of SV’s in the human genome, which can lead to a better understanding of the molecular, biochemical, and cellular events that govern tumor progression.
As Director of the Grandomics Genome Institute, Min works with a talented group of scientists and technologists who develop new genomic solutions to enhance the strengths of the Oxford Nanopore platform for genome science and genome medicine. His team integrates existing and new methods to create a comprehensive pipeline to produce complete animal and plant genomes with a minimum number of gaps. His team also studies the origin, mechanisms, and roles of SV’s in adaptive evolution, complex diseases, and tumorigenesis.
Institute of Molecular & Cellular Pharmacology
Long-read NGS guided preimplantation genetic testing for chromosomal structural rearrangement
Dr Chan graduated from the University of Newcastle Upon Tyne in the UK and gained his PhD at the University of Hong Kong. His research interest is in genetics and epigenetics of hereditary cancers as well as clinical genetic screening. Dr Chan has long been using cutting-edge technologies in his research, including the early application of pyrosequencing in quantification of DNA methylation, leading to the discovery of the mechanism of transcriptional read-through as the cause of Lynch Syndrome. He has been using an NGS approach in preimplantation genetic testing (PGT) since 2015 and his latest work involves the use of long-read NGS for structural variants.
Long-read sequencing and assembly of a large environmental blaCTX-M-15 harbouring plasmid
Infections caused by antimicrobial resistant bacterial pathogens are fast becoming an important global public health issue. Using next generation sequencing data of whole sediment and cultured fractions, our research group have identified wastewater treatment plants (WWTPs) as hotspots for the dissemination of antimicrobial resistance genes/bacteria (ARG/ARB) into the environment. Whilst WWTPs can remove up to 99.9% faecal coliforms, our results suggest that anaerobic digestors and the water treatment process positively select for ARG/ARB. The persistence of plasmid mediated ARGs outside of the host-associated system may play a compounding role in shaping the community-acquired resistome. The aim of our research is to understand the mechanisms of enzyme secretion in E. coli and determine why only ESBLs are in the exoproteome and no other beta-lactamases. Here we investigated the secretory mechanism of an ESBL-producing E. coli strain ST131 isolated from a UK water system. Strains of E. coli ST131 carrying multiple resistance genes, including blaCTX-M-15 (encoding extended spectrum beta-lactamase, ESBL), were isolated from the rivers downstream of WWTPs. We then quantified survival under prolonged anaerobic digestion in the presence and absence of selective antibiotics. We also confirmed if the gene was plasmid borne and studied the secretory mechanisms associated with all beta-lactamases in the genome. Here we present a method to rapidly sequence, assemble and undertake primary annotation of the bla genes carrying plasmid that are associated with our environmental E. coli ST131. The use of Oxford Nanopore long-read sequencing has permitted accurate de novo assembly and has helped further resolve the AMR genes location, composition, order, function and putative mechanism of transposition. Such assembly has been previously unachievable using our existing short-read sequence data set.
Séverine Rangama is currently a PhD student at the School of Life Sciences, University of Warwick. Her research aims to develop an increased understanding of beta-lactam resistance gene expression and to elucidate the secretion of the enzyme via the SecA pathway.
Long-read sequencing technologies resolve most dark and camouflaged gene regions
Complex genomes, including the human genome, contain ‘dark’ regions that standard short-read sequencing technologies do not adequately resolve, including protein-coding genes, leaving many variants that may be relevant to disease entirely overlooked. We systematically identified gene regions that are ‘dark by depth’ (few mappable reads), and others that are ‘camouflaged’ (ambiguous alignment). More than 100 protein-coding genes are 100% camouflaged using standard short-read sequencing. Many known disease-relevant genes are also camouflaged, including CR1, a top Alzheimer’s disease gene, and other disease-relevant genes include NEB, SMN1 and SMN2, and ARX. We further assessed how well long-read technologies resolve these regions, including 10x Genomics, PacBio’s Sequel, and Oxford Nanopore PromethION (Cliveome v. 3.0). We found that long-read technologies largely resolve the camouflaged gene regions, making it possible to identify mutations that may be important in human disease.
Dr. Ebbert is an Assistant Professor of Neuroscience at the Mayo Clinic with a background in computational biology and bioinformatics, focusing on Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD). He also has experience in genomics studies and analyses, algorithm design, and statistics. He has published in respected journals across cancer, bioinformatics, and Alzheimer’s disease, and recently published a manuscript demonstrating that long-read technologies can traverse the challenging C9orf72 ‘GGGGCC’ repeat expansion.
Measuring transcriptomic diversity induced by genome SCRaMbLEing with nanopore direct RNA sequencing
Dr Aaron Brooks earned his PhD from the University of Washington and is currently an EMBL Interdisciplinary Postdoc (EIPOD) working with Dr Lars Steinmetz in Heidelberg, Germany. Aaron’s research harnesses synthetic and evolutionary biology to understand how the physical layout of the genome shapes its function. Aaron and his team have relied on nanopore sequencing to detect abrupt reorganization events in synthetic genomes and measure their consequences.
Mobile Malaria project
Oxford Nanopore Technologies
Dan Turner is Vice President of Applications at Oxford Nanopore Technologies and is a highly experienced scientist who has worked in the field of next-generation sequencing for the last 11 years. Dan provides scientific leadership for multi-disciplinary teams in Oxford, New York and San Francisco. The Applications group aims to bring together sample prep technologies, genomics applications and bioinformatics, to expand the utility of Oxford Nanopore Technologies devices and illustrate the benefits of these technologies to the wider world. Before joining Oxford Nanopore Technologies, Dan was Head of Sequencing Technology Development at the Wellcome Trust Sanger Institute, and prior to this he held postdoctoral positions at the Sanger Institute and Cornell University Medical College in Manhattan.
Oxford Nanopore Technologies
Eoghan Harrington is the Associate Director of Genomic Applications Bioinformatics 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 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.
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.
Redefining the transcriptional complexity of viral pathogens using direct RNA sequencing
Revealing mRNA alternative splicing complexity in the human brain
Nicola Hall is a postdoctoral researcher at the University of Oxford in Department of Psychiatry with the Tunbridge group. She is using her background in molecular biology and RNA sequencing to investigate gene expression in the human brain. Her current work focuses on alternative splicing of the calcium channel CACNA1C, implicated in schizophrenia and bipolar disorder. Nicola completed her PhD in 2017 at the University of Oxford in the Department of Biochemistry.
RWTH Aachen University
Ship-Seq: nanopore sequencing of polar microbes onboard research vessels
One of the exciting features of the MinION is the ability to carry out in situ sequencing in remote environments where previously it has been impossible to effectively study ecosystems. Polar oceans are biodiversity hot-spots which disproportionately contribute to global biogeochemical cycles, but they are among the most under-explored ecosystems on Earth, as well as the most threatened by anthropogenic environmental change. As a result of this, there is increasing interest in the study of polar microorganisms such as diatoms and coccolithophores, which are the main regulators of the polar ocean biogeochemical cycles. The study of polar microbes is often challenging as they survive only at specific temperatures, which limits our ability to transport them to laboratories for experiments. Long-term maintenance in the laboratory is also problematic as many species are cold-adapted and require polar-specific environments, as well as failing to thrive in close quarters. We are addressing this challenge by using the MinION for real-time studies on the diversity and function of microbial communities from the surface ocean. Our aim is to provide a real-time assessment of microbial diversity, real-time analysis of in situ experiments in polar oceans, and genome and transcriptome sequencing of sensitive but ecologically relevant polar microbes. During January and February 2019, we carried out our first feasibility test during a research cruise on the RRS Discovery, in which a MinION was used in conjunction with NanoOK RT software for in situ sequencing and real-time analysis of metagenomic samples collected by the ship. Our experiment provided information about species composition and abundance at multiple sampling points on a long transect between the Falkland Islands and South Georgia and the South Sandwich Islands, crossing the polar front. This includes a range of nutrient levels and temperatures, which allows for the investigation of genetic basis for the ability of diatoms and other phytoplankton to survive in a wide variety of conditions. Real-time analysis onboard a research vessel allows researchers to make evidence-based decisions on sampling locations and whether sampling has been sufficient. The results from our experiment will be validated against previous data from similar locations, alongside sequencing of sample replicates using alternative platforms. This will allow for a comparison between in situ sequencing with the MinION and UK-based sequencing with other platforms. Our results indicate that MinION sequencing is a powerful tool for polar microbe research, although a lack of available reference genomes currently limits its power. For further investigations, alongside the production of more reference genomes, analysis pipelines will be tailored to target specific genes and species that are of interest in terms of their function and ecological role.
Emma Langan is a NEXUSS PhD student at the University of East Anglia, Earlham Institute and the British Antarctic Survey, where she is using the Nanopore MinION for real-time metagenomic sequencing of polar ocean samples to monitor microbe populations. Emma graduated from University of Edinburgh with a BSc in Biomedical Sciences (Infectious Diseases) in 2015, before completing a MSc in Bioinformatics where she built a genome browser for the investigation of silent cricket genotypes.
Splice isoform-specific analysis of endogenous NMD targets in human cells
Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that targets mRNAs with premature termination codons, as well as some endogenous mRNAs that encode full-length proteins. The features that render an mRNA sensitive to NMD are still poorly understood, except for the presence of an exon junction complex (EJC) >55 nts downstream of the termination codon. Obscuring the identification of NMD-inducing features is the fact that previous transcriptome-wide analyses of endogenous NMD targets did not reveal which specific splice isoforms are degraded by NMD. This is mostly attributed to the insufficient coverage of splice junction sites and the lack of information regarding non-annotated mRNA isoforms that are enriched upon NMD inhibition. A recent comparative transcriptome analysis from our lab of cells, in which three essential NMD factors were knocked down and then rescued, identified a high-confidence set of genes whose transcripts react to NMD (Colombo et al., RNA, 2016). However, because the analysis was based on short-reads only, we could not obtain reliable isoform-specific information. For an isoform-specific analysis, we now use cDNA nanopore sequencing, which allows us to identify full-length mRNAs that are stabilized upon NMD inhibition. Our approach can detect full-length isoforms that are enriched, or even appear, when NMD is inactivated and we have experimentally verified several examples. We integrate long and short-read sequencing to accurately quantify the expression of individual isoforms and thereby identify those that are targeted by NMD. We aspire to reveal the regulatory role of NMD at isoform-specific level and generate a resource that will enable the study of features that render a specific mRNA sensitive to NMD.
Evangelos is a postdoc in Oliver Mühlemann’s group at the Department of Chemistry and Biochemistry, University of Bern, Switzerland. He is interested in post-transcriptional mRNA regulation in mammalian cells and applies nanopore sequencing to identify endogenous mRNAs that are sensitive to nonsense-mediated mRNA decay. He is a biochemist from Greece with background in transcriptomics, translation termination and RNA decay.
Tapestry: assessing small eukaryotic genome assemblies with long-reads
Assemblies of small eukaryotic genomes using long-reads are often close to complete. However, these assemblies remain difficult to validate, especially when genomes have complex features such as large inversions, translocations, ploidy variations, and where chromosome number may not be known. While many tools for assessing assemblies with short-reads exist, long-reads have far greater power for confirming the accuracy and completeness of contigs. I will present Tapestry, a tool for validating the contigs of a small assembly automatically and visualising the contigs so the structure of the assembly can be refined before polishing. I will show how Tapestry has helped us to resolve the complex genomes of several small eukaryotes.
John Davey is a bioinformatician at the University of York, working in the Department of Biology Technology Facility. He received his PhD from the University of Edinburgh and then worked with Mark Blaxter and Edinburgh Genomics during the development of Illumina sequencing, developing methods for analysing Restriction-site Associated DNA (RAD) Sequencing data, among many other things. He then held a fellowship at the University of Cambridge, working with Chris Jiggins on speciation of Heliconius butterflies, completing a chromosomal genome assembly of H. melpomene. He now works on a wide range of genomes and metagenomes at York, mostly trying to figure out how to turn raw nanopore sequence into completed genome assemblies.
Telomere-to-telomere assembly of a complete human X chromosome
Release of the first human genome assembly was a landmark achievement, and after nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has yet been finished end to end, and hundreds of gaps persist across the genome. These unresolved regions include segmental duplications, ribosomal rRNA gene arrays, and satellite arrays that harbor unexplored variation of unknown consequence. We aim to finish these remaining regions and generate the first truly complete assembly of a human genome.
Here we announce a whole-genome de novo assembly that surpasses the continuity of GRCh38, along with the first complete, telomere-to-telomere assembly of a human X chromosome. In total, we collected 40X coverage of ultra-long Oxford Nanopore sequencing for the CHM13hTERT cell line, including 44 Gb of sequence in reads >100 kb and a maximum read length exceeding 1 Mb. This unprecedented coverage of ultra-long reads enabled the resolution of most repeats in the genome, including large fractions of the centromeric satellite arrays and short arms of the acrocentrics. A de novo assembly combining this nanopore data with 70X of existing PacBio data achieved an NG50 contig size of 75 Mb (compared to 56 Mb for GRCh38), with some chromosomes broken only at the centromere. Using this assembly as a basis, we chose to manually finish the X chromosome. The few unresolved segmental duplications were assembled using ultra-long reads spanning the individual copies, and the ~2.3 Mbp X centromere was assembled by identifying unique variants within the array and using these to anchor overlapping ultra-long reads. These results demonstrate that it is now possible to finish entire human chromosomes without gaps, and our future work will focus on completing and validating the remainder of the genome.
Karen H. Miga, PhD, is an Assistant Research Scientist at UCSC. Dr. Miga’s research program combines innovative computational and experimental approaches to produce the high-resolution sequence maps of human centromeric and pericentromeric DNAs.
The Hong Kong University of Science & Technology
Ultra-long reads and ultra-long duplications: deciphering the mysteries of the Bordetella pertussis genome
In light of widespread resurgence of the respiratory disease whooping cough, ongoing research aims to identify changes to the causative bacterium, Bordetella pertussis. B. pertussis is traditionally described as a highly clonal species at the single-base level, hence our research largely focusses on identifying differences between strains on a whole-genome scale. Long-read sequencing has enabled us to produce closed genome sequences for B. pertussis isolates on an unprecedented scale, allowing visualisation of extensive inter-strain genomic rearrangements. This work also led to the unexpected discovery of a second phenomenon: large duplications which are present in some recent isolates but not in the B. pertussis reference genome. Intriguingly, these duplications may be present in only a fraction of the cells of duplication-carrying strains. At London Calling 2019, I will discuss this developing story, including the essential role of long and ultra-long nanopore sequencing in proving the existence of the duplications and characterising variable populations, alongside continuing work to quantify the phenotypic effects of the duplications.
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 of Bordetella pertussis, the bacterium responsible for whooping cough.
Understanding the role of TEs in cellular differentiation at single cell resolution
Transposable elements (TEs) are long known to be expressed in different cells during early mammalian development. However, the role of TEs in cellular differentiation has remained elusive. We have developed a new experimental and computational methods to understand the role of TEs in cellular differentiation at single cell resolution.
Single cell (sc) RNA sequencing (RNA-seq) has been developed extensively in recent years to study cell-to-cell variability of gene expression. These methods, however, have exclusively used short read sequencing technologies, which do not allow for TE mapping. We have developed a novel plate-based long read scRNA-seq protocol, which will overcome this limitation. Full-length transcripts are tagged with unique molecular identifiers (UMIs) prior to amplification, permitting accurate transcript counting. We also introduced PCR barcoding allowing for pooling of samples, this will further decrease the PCR amplification.
We have devised a computational method to error correct reads using UMIs, by calculating a consensus from multiple sequence alignments of all reads flagged as PCR duplicates.
This protocol allowed for the first time for long-read sequencing of scRNA-seq libraries incorporating error correction of Oxford Nanopore reads. We used this method to study transposable element expression in single cells at single molecule resolution in Dicer KO mouse embryonic stem cells.
Rebecca Berrens completed her PhD at Cambridge University where she investigated how expression of non-genic regions of the genome and transposable elements (TEs) are controlled by epigenetic modifications during early development. Rebecca is currently a Research Associate with John Marioni at CRUK Cambridge Institute, where she is developing computational and experimental tools to perform single cell RNAseq using Oxford Nanopore Technologies to study the role of transposable element expression in cellular differentiation.
University of Birmingham
At present, most metagenomic surveys are performed using short-read sequencing. This approach limits the specificity of taxonomic assignment and result in highly fragmented assemblies. Single molecule sequencing platforms are able to sequence much longer molecules and the output of these platforms, particular the PromethION from Oxford Nanopore, now supports the study of complex microbial communities using shotgun metagenomics. We assessed a variety of commercially available and manual extraction methods using both a ten-species mock community and clinical samples of stool to find a method capable of generating ultra-long reads (>100 kb). Neither bead-beating or column-based extraction methods were found to support reads of the desired length and moving to magnetic bead and manual extraction methods allowed significant improvements in read-length. We also demonstrated the power of using solely chemical and enzymatic cell lysis methods for extracting high-molecular weight DNA from recalcitrant organisms, such as Gram-positive bacteria and fungi, over popular physical disruption methods. Development of these methods is critical to support the growing field of clinical microbiome research, including the ability to perform strain tracking and produce high-quality metagenome assembled genomes (MAGs) from metagenomic samples.
University of Cambridge
University of Melbourne
University of Nottingham
Unraveling genomic secrets at sea: Sequencing sharks and microbes on the MinION
Chondrichthyes - sharks, rays and chimaeras (‘sharks’) evolved 500 million years ago and are one of the oldest extant vertebrates today. Sharks have extraordinarily long-life spans, exceptional wound healing capabilities and large genomes – qualities which make them ideal candidates for understanding mechanisms contributing to genome stability and immunological resilience. As apex predators, sharks are also vital to top-down regulation of oceanic ecosystems and are therefore crucial to maintaining commercial fish stocks and human food security. However, sharks are disproportionately targeted to meet the international demand for shark fins and as a result an estimated 25-50% of species are threatened by extinction. 50% of shark species are also data deficient, making it difficult to conserve remaining populations and to study their evolutionary adaptations. Our goal is to reduce data deficiency of shark populations through on-site genomic and metagenomic studies in shark biodiversity hot-spots, including the USA, India, Tanzania, Mexico, Australia, and Philippines. Shark samples are collected from free swimming sharks or from specimens found in fish markets. Genomic DNA is sequenced on-site by trained undergraduate and graduate students on the MinION. We sequenced four new chondrichthyan genomes including the Silky shark (Carcharhinus falciformis), Sharpnose guitarfish (Glaucostegus granulatus), and two manta rays (Mobula japonica and Mobula tarapacana). Long-read sequencing on the MinION allowed high depth of sequencing coverage of shark genomes, which are typically 1-6 gigabases in size. Our studies increased the number of sequenced chondrichthyan genomes by 40%. Ongoing genome assessments for population size and structure will allow determination of conservation status for these shark species. Genome comparisons across taxa will increase understanding of mechanisms which impart evolutionary resilience to this species group. Further, our microbiome analyses of free-swimming whale sharks (Rhincodon typus) in locations across the globe revealed that microbiomes are similar with respect to taxonomic composition and functional profiles in genetically diverse and geographically separated whale shark populations, providing key insights about the biogeography of whale sharks. Analyses of functional profiles of the microbiome in wild thresher sharks (Alopias vulpinus) revealed a10-fold higher proportion of heavy metal-metabolizing genes in sharks as compared to the water column in coastal San Diego, suggesting either bioaccumulation of heavy metals or a novel baseline microbiome specific to thresher sharks. In summary, use of portable sequencing technology from Oxford Nanopore has improved the data deficiency of shark populations through local capacity building and will facilitate greater protection of endangered species in the future.
Untangling heterogeneity in DNA replication with nanopore sequencing
Genome replication is a stochastic process whereby each cell exhibits different patterns of origin activation and replication fork movement. Despite this heterogeneity, replication is a remarkably stable process that works quickly and correctly over hundreds of thousands of iterations. Existing methods for measuring replication dynamics largely focus on how a population of cells behave on average, which precludes the detection of low probability errors that may have occurred in individual cells. These errors can have a severe impact on genome integrity, yet existing single-molecule methods, such as DNA combing, are too costly, low-throughput, and low-resolution to effectively detect them. We have created a method called D-NAscent that uses Oxford Nanopore sequencing to create high-throughput genome-wide maps of DNA replication dynamics in single molecules. I will discuss the informatics approach that our software uses, as well as questions pertaining to DNA replication and genome stability that our method is uniquely positioned to answer.
Michael Boemo is a postdoctoral research assistant in the Sir William Dunn School of Pathology at University of Oxford with Professor Conrad Nieduszynski, and currently holds the Emanoel Lee Junior Research Fellowship at St. Cross College. Michael completed his PhD in condensed matter physics in 2016 at the University of Oxford where, together with Professor Andrew Turberfield and Professor Luca Cardelli, he developed a computing system comprised of autonomous robots made from DNA. Dr Boemo is interested in developing computational methods to study systems biology, and his current work aims to develop methods to study DNA replication dynamics at single-molecule resolution and a new process algebra for the simulation of biological systems.
Using direct RNA sequencing to detect RNA structures in transcriptomes
Yue Wan received her BSc in Cell Biology and Biochemistry from the University of California, San Diego and her PhD in Cancer Biology from Stanford University under the mentorship of Howard Y. Chang. During her PhD, she developed the first high-throughput method for probing RNA structures genome-wide. Yue is a recipient of the NSS-PhD scholarship from the Agency for Science, Technology and Research (A*STAR) in Singapore and is currently a Principle Investigator in the Genome Institute of Singapore. She is a Society in Science- Branco Weiss Fellow, EMBO Young Investigator and CIFAR-Azrieli Global Scholar, as well as a recipient of the Young Scientist Award and L’Oreal-UNESCO for Women in Science, Singapore National Fellowship. She is interested in studying functional RNA structures and understanding their roles in regulating cellular biology.
Using full-length transcript sequencing to reveal the fate of mRNA in aging seeds
Margaret Fleming obtained her PhD in Botany from Colorado State University in 2015 for her work on the role of the structural cell wall protein extensin in biomass recalcitrance in the context of biofuel production. Margaret then completed a postdoc with Dr Christina Walters at the National Laboratory for Genetic Resource Preservation, where she studied how time and environment affect seeds of both cultivated and wild plants, focusing on the interrelationship of RNA degradation and seed viability. Her current work with Dr Chris Saski focusses on the transcriptomic effects of Armillaria (root-rot) infection of susceptible and resistant peach rootstock and will soon join the lab of Dr Marjorie Weber at Michigan State University to study the evolution of mite domatia in Vitis (grape).