Assessing the prevalence and sequence diversity of Hepatitis B Virus in Sierra Leone with MinION
While Sierra Leone has implemented a robust program to combat HIV infection, there is little to no infrastructure in place for surveillance or treatment of other blood-borne pathogens, such as viral hepatitis. In collaboration with Magbenteh Hospital, University of Makeni Infectious Disease Research Laboratory and the University of Cambridge, over the past 24-months we have collected and screened residual, anonymised blood samples for the presence of viral pathogens, including Hepatitis B Virus (HBV). At present, 7384 samples (2620 male, 4764 female), have been collected and screened. Of this cohort, 7137 have been screened for HBV by qPCR, representing a significantly larger cohort than previously screened within Sierra Leone. Our results found 582 PCR positive patients (8.2%), which is very high considering this represents active infections in the community. We sequenced the positive patients using nanopore sequencing technology in country, and assessed the circulating genotypes, prevalence of drug and antibody resistance markers. We found overwhelmingly that genotype E variants are in circulation, while we also observed a wide range of drug and vaccine resistance phenotypes, which is concerning considering the lack of treatment options within the country. This study demonstrates the utility of nanopore sequencing in the field, while also highlighting the problem of HBV in Sierra Leone.
Dr. Luke Meredith is a post-doctoral research fellow based at the University of Cambridge, working with Prof. Ian Goodfellow. Obtaining a PhD in Molecular Virology in 2009, Dr. Meredith's post-doctoral work focused on virus binding and entry. In 2015, he joined the West African Ebola outbreak response and spent 7 months in Sierra Leone, initially as part of the Public Health England diagnostic response, then subsequently as part of the clinical research team evaluating diagnostic tests. He then spent a further 3 months running the Ebola Outbreak Sequencing Service, providing real-time sequencing support to the response, enabling the rapid characterisation of EVD cases during the latter stages of the epidemic. Following the epidemic, Dr. Meredith played a key role in the establishment of the University of Makeni Infectious Disease Research Laboratory, a legacy teaching and research laboratory which provides training to Sierra Leonean graduate students, as well as being the base for research and surveillance projects monitoring the diversity and prevalence of infectious disease in Sierra Leone and surrounding countries in West Africa.
Bacterial species level identification and strain level differentiation using repetitive extragenic palindromic based amplicon sequencing with Oxford Nanopore Technology
We have developed a new method for fast and cost-effective bacterial species level identification and strain level differentiation using Repetitive Extragenic Palindromic based amplicon sequencing on MinION platform. The method utilizes an optimized version of rep-PCR followed by dual-stage rep-PCR-2, during which sample specific barcodes are incorporated. DNA enrichment together with barcoding takes less than 5 hours and ensures highly repetitive and evenly distributed reads per sample. Our results demonstrate that sequencing of the rep-PCR genomic fingerprint profile with Oxford Nanopore Technology generates highly reproducible peak profiles. We have developed a pipeline that, by correcting the random error of individual reads within each peak, generates a set (~10 reads per sample; 300bp - 3Kb) of high quality (>99%) consensus reads. The information from high quality reads is used to retrieve species level identification. Furthermore, we have developed an algorithm that compares integrals of the peaks profiles allowing for strain level discrimination.
Lukasz Krych completed his PhD in Food Microbiology in 2014 at the University of Copenhagen, where he is now a post-doctoral researcher. He utilises molecular genetics and classical microbiological techniques to investigate the association of the gut microbiome with health and disease. This involves the development of novel data analysis tools, multivariate data mining and protocol optimisation. Lukasz is also R&D director at GenXone, a Polish biotech company specialising in the development of diagnostic products based on the latest DNA testing technologies.
Clinical applications for real-time sequencing in leukaemia
Leukemias are characterized by a variety of distinct cytogenetic and molecular subgroups that impact response and survival. Many of these leukemias have specific translocations and mutations that confirm the suspected diagnosis, provide prognostic information, and guide therapy. A particular challenge in current molecular pathology practice is the high cost and long turnaround times for reporting of molecular profiles in these leukemias. Our research aims to use real-time sequencing for more efficient and improved molecular diagnostics workflows. Our work involves development of a real-time sequencing assay for detection of fusion genes and to sequence the FLT3 gene transcript to detect mutations in leukemias using the MinION platform.
Dr. Cecilia Yeung is an Associate Member at the Fred Hutchinson Cancer Research Center where she serves as the medical director of the Molecular Oncology Laboratory. She is an Assistant Professor at the University of Washington, Department of Anatomic Pathology where she serves as the chair of the Clinical Competency Committee for the Molecular Genetics Pathology Fellowship, and the director for the Molecular Pathology Elective. She is the co-coordinator and speaker of the Molecular Pathology Lecture Series for residents and fellows. She is a member of the Board of Directors and the chair of the Teaching and Education Committee at the Association for Molecular Pathology. Her clinical appointment at Seattle Cancer Care Alliance is where she focuses her pathology diagnostic skills in immunotherapy, transplant, and hematopathology. Her research interest focuses on developing novel molecular diagnostics for hematologic malignancies, and improving correlative data from clinical trials via implementation of better translational medicine diagnostic assays.
Dr. Olga Sala Torra is a staff scientist in the Radich laboratory at the Fred Hutchinson Cancer Research Center in Seattle, WA. Her main research interests are the application of gene expression and sequencing techniques to the detection of minimal residual disease in leukemias, and the development of low-cost diagnostic methods for hematologic malignancies that can be useful in low-income countries.
Sala Torra, O. et al. Next-Generation Sequencing in Adult B cell Acute Lymphoblastic Leukemia Patients. Biology of Blood and Marrow Transplantation 23, 691–696 (2017).
Torra, O. S., Beppu, L., Smith, J. L., Welden, L., Georgievski, J., Gupta, K. Radich, J. P. (2016, June 2). Paper or plastic? BCR-ABL1 quantitation and mutation detection from dried blood spots. Blood. American Society of Hematology. https://doi.org/10.1182/blood-2015-12-689059
Complex structural variants resolved by short-read and long-read whole genome sequencing in Mendelian disorders
Complex structural variants (cxSVs) are genomic rearrangements comprising multiple structural variants, typically involving three or more breakpoint junctions. They contribute to human genomic variation and can cause Mendelian disease, however they are not typically considered during genetic testing. We investigated the role of cxSVs in Mendelian disease using short-read whole genome sequencing (WGS) data from 1,324 individuals with neurodevelopmental or retinal disorders from the NIHR BioResource project. We identified four cases of individuals with a cxSV affecting Mendelian disease-associated genes. We used nanopore sequencing to resolve the mechanism of one of the variants. Our results show cxSVs are an important, although rare cause of Mendelian disease, and we therefore recommend their consideration during research and clinical investigations.
Alba Sanchis-Juan studied Biochemistry and Biomedicine at University of Valencia, Spain. She is a PhD student and works at the Department of Haematology, University of Cambridge. Her study has been focused on the use of next-generation sequencing for clinical research and diagnosis. Currently, she performs data analysis of whole genome sequence data of individuals with neurodevelopmental disorders.
Sanchis-Juan, A. et al. Complex structural variants resolved by short-read and long-read whole genome sequencing in Mendelian disorders. bioRxiv 281683 (2018). doi:10.1101/281683
De novo sequencing and assembly of plant genomes using nanopore long reads
Plant genomes are often characterized by their high level of repetitiveness and polyploid nature. As a direct consequence, genome assemblies of plant genomes are challenging. The introduction of short-reads technologies ten years ago, significantly increased the number of available plant genomes. Generally, these assemblies are incomplete and fragmented, and only a few of them are at the chromosome-scale. Recently, Oxford Nanopore sequencing technology was commercialized with the promise to sequence long DNA fragments (kilobases to megabases order) and then, by using efficient algorithms, provide assembly of high quality in terms of contiguity and completeness of the repetitive regions. Here we describe the de novo sequencing and assembly of several plant genomes (banana, citrus and brassicaceae) and the impact of read length on the contiguity and completion of genome assemblies.
Jean-Marc Aury is a researcher at Genoscope since 2003. He was focused on eukaryotic genome analysis and was a main actor of several genome projects, like paramecium, grape, banana, cocoa and oak. He is now the team leader of a bioinformatic group which is focused on sequencing data production, genome assembly and gene prediction in eukaryotic genomes, with a broad interest in methodological development. Genoscope acts as the French National Sequencing Centre since 1998 and has an extensive experience in large sequencing projects (for example the Tara Oceans metagenomic project). Genoscope has participated in the MinION and PromethION Early Access Program.
Istace, B. et al. de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. GigaScience 6, (2017).
Schmidt, M. H. et al. de novo assembly of a new Solanum pennellii accession using nanopore sequencing. The Plant Cell tpc.00521.2017 (2017). doi:10.1105/tpc.17.00521
Carradec, Q. et al. A global ocean atlas of eukaryotic genes. Nature Communications 9,(2018).
Madoui, M. A. et al. Genome assembly using nanopore-guided long and error-free DNA reads. BMC Genomics 16, (2015).
Detecting DNA modifications and structural variants from nanopore long-read sequencing data
Compared to short-read sequencing techniques, Oxford Nanopore Technologies long-read sequencing platform has several advantages, such as the ability to detect DNA modifications directly from electric signals, and the improved sensitivity to find structural variants. Here we describe a novel method called NanoMod to improve the performance of detecting DNA modifications, especially synthetically introduced modifications, by analyzing the characteristics of raw signal intensities. We also describe a data handling pipeline for detecting structural variants from long-read data. We illustrate a few examples of pinpointing the exact breakpoints of balanced translocations or identifying causal structural variants in exome-negative patients, which subsequently enabled pre-implementation genetic diagnosis.
Dr. Kai Wang is an Associate Professor at the Raymond G. Perelman Center for Cellular and Molecular Therapeutics of the Children’s Hospital of Philadelphia, and Department of Pathology & Laboratory Medicine at the University of Pennsylvania’s Perelman School of Medicine. He received a Bachelor’s degree from Peking University in China, a master’s degree from the Mayo Clinic, and a PhD from the University of Washington. He had postdoctoral training at the University of Pennsylvania and the Children’s Hospital of Philadelphia, before becoming an Assistant Professor, and later Associate Professor at the University of Southern California Keck school of Medicine, and then Columbia University Medical Center. His research focuses on the development and application of genomic approaches to study the genetic basis of human diseases and facilitate the implementation of genomic medicine.
Liu, Q., Georgieva, D. C., Egli, D. & Wang, K. NanoMod: a computational tool to detect DNA modifications using nanopore long-read sequencing data. bioRxiv 277178 (2018). doi:10.1101/277178
Detection of clinically relevant molecular alterations in CLL by nanopore sequencing
Chronic Lymphocytic Leukaemia (CLL) is the most common form of leukaemia in the Western world, and is characterised by both clinical and biological heterogeneity. The majority of CLL patients display few symptoms at diagnosis, after which the disease can progress into either an aggressive, chemo-resistant form with poor prognosis, or a relatively indolent form with a life expectancy similar to that seen in the normal population. The path taken by any particular CLL case is influenced by the presence or absence of a number of specific molecular alterations, including copy number changes such as trisomy 12, del(11q), del(13q) and del(17p), the mutational status of the immunoglobulin heavy chain (IgHV), and the presence of somatic mutations within TP53. As such, identification of these changes is an important part of the treatment decision process. Here I will highlight our work using a combination of targeted and ultra-low coverage whole genome sequencing on the MinION platform, to simplify the detection of these clinically important molecular alterations in CLL.
Dr Adam Burns is a post-doctoral researcher in the Oxford Molecular Diagnostics Centre based in the Department of Oncology at the University of Oxford. His research focuses on developing next-generation sequencing-based tools for clinical diagnostic use. After obtaining his BSc from the University of Hull, Adam worked in industry for five years before joining Oxford University in 2009. There he developed screening assays for clinically relevant mutations, including KRAS, BRAF and JAK2, across a range of haematological malignancies. As part of his PhD from Oxford Brookes University in 2016, Adam developed targeted and whole genome sequencing approaches to help diagnose haematological malignancies. This work was subsequently adopted for routine diagnostic use in the Oxford University Hospitals NHS Foundation Trust and also informed the Genomics England 100,000 Genomes Project. Adam’s current research is centred on using nanopore sequencing, and other patient-near technologies, as an accurate, low-cost, easy-to-use screening platform to detect clinically relevant genetic changes in haematological malignancies for use in resource-poor regions of the world.
Burns, A. et al. Whole-genome sequencing of chronic lymphocytic leukaemia reveals distinct differences in the mutational landscape between IgHVmut and IgHVunmut subgroups. Leukemia 32, 332–342 (2018)
Stamatopoulos B, et al. Targeted deep sequencing reveals clinically relevant subclonal IgHV rearrangements in chronic lymphocytic leukemia. Leukemia 31, 837–845 (2017)
Pellagatti, A. et al. Targeted resequencing analysis of 31 genes commonly mutated in myeloid disorders in serial samples from myelodysplastic syndrome patients showing disease progression. Leukemia 30, 247–250 (2016)
Detection of GBA missense mutations and other variants using the Oxford Nanopore MinION
Mutations in GBA cause Gaucher disease when biallelic, and are strong risk factors for Parkinson’s disease when heterozygous. GBA analysis is complicated by the presence of a nearby pseudogene. Here we present a method for sequencing GBA, using an amplicon including all coding regions and introns, on the Oxford Nanopore MinION, enabling a fast and comprehensive assessment. For illustration we successfully sequenced DNA samples from 17 individuals, including patients with Parkinson’s and Gaucher disease, in a study combining earlier and current nanopore chemistry. We initially compared different aligners (Graphmap and NGMLR), and used Nanopolish and Sniffles to call variants, and NanoOK for quality metrics. Many samples had previously known mutations, including the common p.N409S (N370S) and p.L483P (L444P). We detected these, mostly in a blinded fashion, and other causative mutations in Gaucher patients. In a sample with the complex RecNciI allele, we detected an additional coding SNP, and a 55-base pair deletion in data aligned by NGMLR. We haplotyped all samples using Whatshap and confirmed compound heterozygosity where relevant. False positives were fewer with NGMLR, and easily identified and filtered. We demonstrate the potential of the MinION to analyse this difficult gene, with the added advantage of phasing and intronic analysis.
Christos Proukakis is a clinical academic neurologist, holding a senior lectureship at UCL Institute of Neurology and is an honorary consultant neurologist at the Royal Free NHS Trust. His work focuses on Parkinson’s disease, investigating the role of somatic mutations and the utility of new sequencing technologies.
Nacheva, E. et al. DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation. PLoS ONE 12, (2017)
Kara, E. et al. Genetic and phenotypic characterization of complex hereditary spastic paraplegia. Brain 139: 1904-18, (2016)
Kiely, AP. et al. Distinct clinical and neuropathological features of G51D SNCA mutation cases compared with SNCA duplication and H50Q mutation. Mol Neurodegener,10:41, (2015)
Porcari, R. et al. The H50Q mutation induces a 10-fold decrease in the solubility of α-synuclein. Journal of Biological Chemistry 290, 2395–2404 (2015)
Beavan, M. et al. Evolution of prodromal clinical markers of parkinson disease in a GBA mutation-positive cohort. JAMA Neurology 72, 201–208 (2015).
Determining novel antimicrobial resistance mechanisms with Oxford Nanopore long-read sequencing data
Next-generation sequencing technology has revolutionized the study of microbial genomics and has the potential to enable near real-time pathogen identification and antimicrobial resistance pattern prediction in clinical settings. The use of traditional short-read sequencing has limitations, as it is difficult to resolve repeats, multiple replicons, and other complex antimicrobial resistance mechanisms in the analysis of single bacterial genomes. The use of Oxford Nanopore sequencing overcomes these limitations, allowing us to leverage ultra-long reads to resolve these complex resistance mechanisms and identify new and novel ones as well. I will present the work our lab is conducting to elucidate complex resistance mechanisms, including a large tandem duplication of a b-lactamase encoding transposon in an Escherichia coli plasmid, the duplication of a KPC-3 encoding transposon within a large plasmid in Klebsiella pneumoniae, and the resolution of a transposon in a pair of Pseudomonas aeruginosa isolates, all of which provide resistance to antibiotics commonly used to treat infections caused by these bacteria. I will also discuss what our lab is doing to screen for more of these complex resistance mechanisms, with the goal of expanding our understanding of the full repertoire of mechanisms utilized by bacteria, and bringing us closer to near real-time prediction of antimicrobial resistance in clinical settings.
Dr. Hanson is currently an Assistant Professor in the Department of Epidemiology, Human Genetics and Environmental Sciences in the School of Public Health, and the Department of Internal Medicine, Division of Infectious Diseases. He also serves as the Associate Director of Microbial Genomics in the Center for Antimicrobial Resistance and Microbial Genomics (CARMiG) at the McGovern Medical School.
Dr. Hanson’s research interests are in infectious disease transmission and colonization, how microbial communities impact the development of disease, and how antimicrobial resistance develops and transmits through society. He uses a combination of existing and innovative laboratory techniques, as well as cutting-edge sequencing and bioinformatics within his lab’s research.
Evaluating the extensively drug-resistant Klebsiella pneumoniae resistome via MinION direct RNA sequencing
Klebsiella pneumoniae is one of the leading causes of nosocomial infections, frequently possesses multidrug resistance and subsequently results in high mortality. The DNA and RNA of extensively drug-resistant (XDR) K. pneumoniae clinical isolates (Hygeia General Hospital, Greece) were sequenced in this study. MinION sequencing was utilised to assemble these genomes, discern the differential expression of antibiotic resistance genes and ascertain the time required for detection. DNA sequencing identified the majority of acquired resistance (≥75%) resided on plasmids and detected ≥70% of these genes within 2 hours. Direct RNA sequencing successfully revealed aminoglycoside, beta-lactam, trimethoprim and sulphonamide resistance within 2 hours. Resistance towards other antibiotic classes was detected; however, this was dependent on the level of expression which was further validated via qRT-PCR. MinION sequencing was capable of detecting antibiotic resistance in these XDR K. pneumoniae isolates within hours and differential gene expression was successfully discerned via direct RNA sequencing.
Miranda Pitt completed her undergraduate studies at The University of Queensland, Australia. Courses selected in her Bachelor of Biomedical Science degree predominantly focused on infectious disease, immunology and genetics. In 2013, she commenced an Honours degree at the University of Queensland Diamantina Institute, where she investigated differential gene expression in an ankylosing spondylitis mouse model and received an Honours Class I. She is currently a PhD student at the Institute for Molecular Bioscience at The University of Queensland. Her research delves into understanding the mode of action of last resort antibiotics against multidrug-resistant gram-negative bacteria and the genetic basis underpinning resistance.
Pitt, ME. et al. Multifactorial chromosomal variants regulate polymyxin resistance in extensively drug-resistant Klebsiella pneumoniae. Microb Genom (ahead of print) (2018) doi: 10.1099/mgen.0.000158.
Evaluation of nanopore sequencing for bacterial biothreat pathogens
In the event of a deliberate release of a bacterial biothreat agent, rapid characterization of the implicated strain(s) will be critical to the public health response. High quality, whole genome sequencing (WGS) can rapidly reveal genetic engineering, such as the introduction of antimicrobial resistance factors and/or plasmids. Laboratory work with these bacterial pathogens often occurs in space-limited, high containment facilities. On-site nanopore sequencing that produces rapid WGS data with real-time analysis capabilities may reduce the time to results during a public health emergency. Here we present an evaluation of DNA isolation methods and the performance of rapid sequencing library preparation methods for Bacillus anthracis and Yersinia pestis to determine (1) DNA quantity and quality, (2) if the purified DNA can be used for rapid library preparation, (3) the sequence quality of MinION versus Illumina and PacBio data, and (4) whether known antimicrobial resistance markers and plasmids can be identified.
Amy Gargis is a microbiologist at the U.S. Centers for Disease Control and Prevention (CDC), Atlanta. Dr. Gargis earned her M.S. and Ph.D. in Microbiology from The University of Alabama. After receiving her Ph.D. in 2010, she joined the Division of Laboratory Systems at the CDC, where she focused on efforts to improve the quality of genetic testing in the clinical and public health laboratory setting. Dr. Gargis was co-lead in organizing two national workgroups of experts to review and establish consensus guidelines for scientific principles, clinical laboratory practices, regulatory requirements, and professional standards for next-generation sequencing. In 2014, Dr. Gargis joined the BioDefense Research and Development Laboratory within the Division of Preparedness and Emerging Infections at the CDC. In this position, she works to develop and optimize rapid assays to characterize biological threat agents, with an emphasis on detection of antibiotic resistance.
Gargis, A. S., Kalman, L. & Lubin, I. M. Assuring the quality of next-generation sequencing in clinical microbiology and public health laboratories. Journal of Clinical Microbiology 54, (2016)
Gargis, A. S. et al. Good laboratory practice for clinical next-generation sequencing informatics pipelines. Nature Biotechnology 33, 689–693 (2015)
Gargis, a S. et al. Assuring the quality of next-generation sequencing in clinical laboratory practice. Nature Biotechnology 30, 1033–1036 (2012)
Genome assembly in Drosophila using nanopore
The Drosophila genus is a highly-studied group in which many species possess a well-developed set of genetic tools, and for which high-quality genome assemblies are available. This has facilitated studies of function and evolution of cis-regulatory regions and proteins by allowing comparisons across at least 50 million of years of evolution. Yet, there remains substantial genetic diversity within the Drosophila genus that available genomes fail to capture. We asked if Oxford Nanopore technology could be used to rapidly and inexpensively sequence and assemble the genome from any Drosophila species. This technology allowed us to generate high-quality genome assemblies of 16 Drosophila species, including from 11 of the 12 originally sequenced Drosophila species. Alignment of contigs from the published reference genomes to our assemblies demonstrated that approximately 60% of gaps present in currently published reference genomes could be closed using this technology. Importantly, we were able to show that Drosophila assemblies could be generated for approximately $1,000 (USD), providing a roadmap for the affordable sequencing and assembly of additional Drosophila genomes.
Danny Miller received both an MD and PhD from the University of Kansas and completed his PhD work in the laboratory of Scott Hawley at the Stowers Institute for Medical Research. He has always been interested in sequencing more species of Drosophila and understanding structural variation within the melanogaster species group and jumped at the chance to sequence and assemble multiple Drosophila genomes using nanopore technology. He will begin a combined residency in Pediatrics and Medical Genetics at the University of Washington and Seattle Children’s Hospital in June 2018.
Miller, D. E., Staber, C., Zeitlinger, J., & Hawley, R. S. (2018). High-quality genome assemblies of 15 Drosophila species generated using Nanopore sequencing. BioRxiv, 8601, 267393. https://doi.org/10.1101/267393
Solares, E. A., Chakraborty, M., Miller, D. E., Kalsow, S., Hall, K. E., Perera, A. G. Hawley, R. S. (2018). Rapid low-cost assembly of the Drosophila melanogaster reference genome using low-coverage, long-read sequencing. BioRxiv, 267401. https://doi.org/10.1101/267401
How MinION sequencing is helping us solve real-life environmental biotechnology issues
I will be talking about two cases whereby MinION sequencing has provided the insight we need to take environmental biotechnology a step further: Firstly, a landfill is leaching the pesticide Mecoprop into the groundwater. No degradation occurs naturally and the pesticide is therefore gradually spreading. We performed degradation tests and MinION sequencing to identify which bacteria are able to degrade Mecoprop. This information will be used to perform a pilot bioremediation project on-site to remove the pollutant from the groundwater altogether. Secondly, wastewater treatment plants (WWTP) form a crucial barrier between human activities and the environment. Micro-organisms are, for the main part, responsible for degrading hazardous compounds in wastewater, yet very little is known about these wastewater workhorses and how they might be optimised. We are screening the microbiology present in different WWTP’s, together with an industrial water company, to determine the microbial baseline in their treatment plants and which microbial indicators provide early warning that they are not performing optimally. In the longer term this data will be used to determine how wastewater treatment processes can be made more effective.
Aleida is a molecular microbiologist who has been working within the field of environmental and industrial microbiology since 2006 after she graduated from the University of Groningen in the Netherlands. She has always been involved in making complex microbial processes more visible and tangible, for clients and/or own research, through monitoring and measuring using molecular microbial methods e.g. for biological soil remediation, water treatment and biocorrosion. Since May 2016 her company Orvion has started to work with the MinION to provide fast, flexible and in-depth insight into the microbial processes they work with (or against…).
Improving MinION read accuracy to enable the high-throughput analysis of single cell transcriptomes.
We have previously shown that Oxford Nanopore Technologies cDNA sequencing can uncover transcript isoform diversity in single cells in unprecedented detail. Now, we have developed a new method to increase the accuracy of cDNA reads generated by the Oxford Nanopore Technologies MinION. These more accurate reads can be used to demultiplex high-throughput single cell cDNA libraries and identify base-accurate full-length transcript isoforms. We have benchmarked our new method by analyzing a cDNA pool of 96 B cells and found isoform diversity with potential implications for cancer treatment.
Chris Vollmers graduated with a M.S. in Biomedical Sciences from the University of Wuerzburg in Germany before pursuing a Ph.D. through a shared program between the University of Heidelberg in Germany and the Salk Institute for Biological Studies in La Jolla, California. He then joined the lab of Stephen Quake at Stanford University as a postdoctoral fellow and worked on developing genomic tools to analyze B cells on population and single cell levels. He now continues this work in his own lab in the Biomolecular Engineering Department at UCSC since 2014.
Byrne, A. et al. Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. Nature Communications 8, (2017)
Interactive exploration of base calls in nanopore data
Oxford Nanopore Technologies MinION DNA sequencer provides users with large amounts of genomic data in real-time. In addition to the sequence itself, detailed event information is available that includes signal trace, time, and model state of each nanopore. While there are sequencing errors associated with nanopore sequencing, there are not interactive ways for users to assess specific base-pair positions in terms of whether sequence variation corresponds to technical error or true biological diversity. We have developed a REST-API that provides efficient retrieval of data from nanopore FAST5 data. This API has been connected with a front-end visualization extending Pileup.js and making use of Data Driven Documents (D3.js). Together the API and front-end enable a visualization platform for interactive analysis of nanopore signal data to confirm sequence base calls and this software is suitable for use in a mobile setting.
Matthew Links is an Assistant Professor in the Department of Animal and Poultry Science and an associate member of the Computer Science Department at the University of Saskatchewan. His interests in the microbiome have involved numerous contexts: enhanced oil recovery, the rhizosphere, infectious diseases as well as plant and animal health. In these settings Dr. Links’ work has demonstrated that cpn60 is a robust DNA barcode that can be used in microbial profiling studies and provides significant advantages over other genes commonly used for microbial profiling. Recent work has resulted in a novel enrichment technique for microbial profiling (Capture-Seq) that eliminates the need for universal PCR and dramatically reduces the amount of shotgun sequencing data required to gain quantitative information about microbiota from any ecological niche. Matthew’s current work with nanopore sequencing is focused on allowing researchers to interrogate the signal data directly. A key goal of his work is to enable visualization of signal event data in a way that allows a researcher to assess whether a base call was in error, whether the sample was actually from a mixed sample, or represents a heterozygote.
Albert, A. Y. K. et al. A study of the vaginal microbiome in healthy Canadian women utilizing cpn60-based molecular profiling reveals distinct Gardnerella subgroup community state types. PLoS ONE 10, (2015).
Links, M. G. et al. Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds. New Phytologist 202, 542–553 (2014).
Links, M. G., Chaban, B., Hemmingsen, S. M., Muirhead, K. & Hill, J. E. mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences. Microbiome 1, 23 (2013).
Links, M. G., Dumonceaux, T. J., Hemmingsen, S. M. & Hill, J. E. The Chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data. PLoS ONE 7, (2012).
Know your onion - the impact of long reads on large genomes
Of over the > 300K plant species, approximately 500 are domesticated for use by humankind, although 90% acreage is just occupied by 20 species. However, this indicates that there is quite a diversity of genomes, for which reference genome sequences have been developed. Reference genomes have a wide range of sizes and complexity in terms of repeats and ploidy. From the work on the human genome, it has been shown that long-read technologies, such as the one offered by Oxford Nanopore Technologies, can span and resolve complex sequences which previously could not be assembled. In this presentation, I plan to show the power of long nanopore reads to resolve complex puzzles in plant species and the potential to develop completed platinum standard reference genomes in any of the 500 domesticated plant species. For example, improving the assembly of the 16 Gb highly repetitive genome of the onion (Allium cepa).
Richard Finkers graduated in plant breeding working on the identification of loci contributing to resistance to Botrytis cinerea in the tomato. He now leads a research group which focuses on genomics approaches and big data strategies to improve breeding research. The genomics approach mainly seeks to understand the structure and genetic diversity in large and complex crops, for example the development of methodologies to phase sequence reads in the onion (16Gb diploid outbreeding crop) and the four expected phases in the potato (autotetraploid outbreeding crop). Research in big data focuses on strategies to make heterogeneous types of research data FAIR (findable, accessible, interoperable and re-usable) and as input for novel strategies to develop the crops needed for the future.
Wilkinson, M. D. et al. The FAIR Guiding Principles for scientific data management and stewardship. Scientific Data 3, 160018 (2016)
Aflitos, S. et al. Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing. Plant Journal 80, 136–148 (2014)
Motazedi, E., Finkers, R., Maliepaard, C. & de Ridder, D. Exploiting next-generation sequencing to solve the haplotyping puzzle in polyploids: a simulation study. Briefings in Bioinformatics bbw126 (2017). doi:10.1093/bib/bbw126
LOGAN: Lossless graph-based analysis of NGS datasets
Recent developments in sequencing technologies necessitate matching improvements in bioinformatics tools to effectively utilize it. Existing tools suffer from limitations in both scalability and applicability, which are inherent to their underlying algorithms and data structures. We therefore developed a new data structure, the LOGAN graph, which is based on a memory efficient Sparse De Bruijn Graph, with routing information. Unlike the established Overlap-Layout-Consensus or De Bruijn Graph approaches, this LOGAN approach is suitable for both short-read and long-read sequencing data. Here we present the latest results of applying this approach to short-read, long-read and hybrid datasets.
Anthony Bolger completed a BSc in Computing Applications at Dublin City University in 1997, before starting his working career as a consultant. He later set up a software consulting company with a fellow graduate, and after a successful 6 years, relocated to Madrid to pursue an industry position where he served as a Product Architect. Interested in the challenges posed by the rapidly-developing bioinformatics field, Anthony began working in the Max Planck Institute for Plant Physiology in Potsdam in 2009, first as a computational biologist and then as a PhD researcher. He then moved to RWTH Aachen in Germany where he currently works as a post-doc and bioinformatics team lead.
Measuring the transcriptome of the C. elegans lifecycle using direct RNA sequencing
Though regulation of gene expression is central to organism development and disease, there are many open questions about the dynamic nature of gene regulation through development. Here, we take advantage of direct RNA nanopore sequencing (dRNA-seq), to explore some aspects of transcriptome regulation, including splicing variation, and alternative polyadenylation. We have generated transcriptomes for C. elegans, a classic biological model for the study of development, across developmental stages (L1-L4, YA, GA) of the N2 strain.
C. elegans is a relatively simple metazoan with a fully sequenced genome, a well characterized and invariant cell lineage, and an excellent molecular genetic toolbox. It is an ideal system for exploratory development of new genomic technologies, such as the full-length transcript sequencing we propose here. By sequencing full-length transcripts across development, we provide a first look at identification of the diversity and transcript architecture of the transcriptome.
We assessed the prevalence of different splicing and 3’UTR isoforms across our samples, initially focusing on a few genes. In addition, we used a hidden markov model, implemented as part of the nanopolish suite, to estimate poly-A tail lengths, and compare lengths across samples and isoforms.
Nathan Roach is a PhD candidate in the labs of James Taylor and John Kim at Johns Hopkins University in the Cellular Molecular and Developmental Biology department.
MinION in the marine environment: from identifying tetrodotoxin producers to tracing sharks and rays using eDNA
We study marine ecology using molecular biology. In recent years, every summer the neurotoxic Tetrodotoxin (TTX) is found in Dutch mussels and oysters, exceeding levels allowed for human consumption. TTX is produced by bacteria, and accumulates in the animal host. The gene cluster encoding TTX biosynthesis is unknown, and the microbial source of the Dutch TTX outbreak is not identified. We therefore isolated metagenomic DNA from TTX positive shellfish and used nanopore sequencing to identify TTX producing microorganisms and their TTX biosynthetic gene clusters. In another project we survey marine biodiversity. Offshore wind farms in the North Sea are assumed to attract large marine animals, since they provide a diverse habitat with increased biodiversity and shelter. Using a mobile sequencing laboratory, we aim to amplify and sequence environmental DNA (eDNA) directly on-site, to investigate the presence of large marine animals such as sharks and rays. Both applications demonstrate the opportunities of mobile, real-time, long-read sequencing enabled by the MinION.
Reindert Nijland is an Assistant Professor at the Marine Animal Ecology group at Wageningen University, Netherlands. Since obtaining his PhD in molecular microbiology at University of Groningen, Netherlands, he has studied the interaction of bacteria with a diversity of hosts. At the Dove Marine Laboratory, Newcastle University, UK, he worked on biofilms of marine Bacillus species isolated from seaweed. After returning to the Netherlands, he studied bacterial pathogens and their interactions with the human and bovine immune systems at UMC Utrecht. In 2014, he again switched host organism, and joined Wageningen University to study the interactions of bacterial pathogens with their plant hosts. Reindert has a strong passion for marine biology, especially crabs. He enjoys scuba diving, underwater photography and underwater filming. In 2017, he could no longer resist the attraction of the marine environment, and joined the Marine Animal Ecology group. His current focus is on the interaction between marine hosts and their microbes. He developed approaches to identify microbial toxin gene clusters in shellfish. He also works on the development of methods for rapid on-site identification of eukaryotes such as crabs, fish and marine mammals by analysing environmental DNA (eDNA) using nanopore sequencing with the MinION.
Nanopore sequencing in clinical diagnostics
Team Nanopore at Viapath Clinical Laboratories at Guy's, St Thomas's and King's College Hospitals are exploring ways in which the rapid production of long-reads on commodity hardware can be exploited to transform Healthcare Genomics. Whilst base call accuracy of single molecule long-reads is not yet as high as for short-read clusters, unambiguous mapping of long reads opens up unique possibilities not accessible to short-read technology. Furthermore HMM tools can readily be applied to improve the signal:noise ratio to diagnostic standards. We will illustrate how one-step tests can identify gene deletions with base-pair accuracy, trinucleotide repeat expansions in single-reads and report haplotypes directly over kilobase ranges.
Graham Taylor was Head of the Regional Genetics Laboratory in Leeds with a long-standing interest in translating genomic technology into diagnostics. In 2006 he led a UK Department of Health Funded project “New genetic diagnostic technologies for consanguineous families at risk of recessive genetic disease” and moved to Cancer Research UK as Director of Genomic Services, where he led an evaluation of next-generation sequencing technology.
In 2009, as Head of the Genomics Translation Unit in Leeds, his team developed methods for diagnostic amplicon sequencing in fixed tissue, copy number variation analysis and streamlined conventional genetic testing using next-generation sequencing. In 2012 he joined the Department of Pathology at Melbourne University as the Herman Professor of Genomic Medicine, Director of the Australian Node of the Human Variome Project and Director of the Victorian Clinical Genetics Laboratories, helping to accredit both laboratories for exome-based clinical diagnostics.
From February 2016 he took up the post of Scientific Director of Clinical Genomics with ViaPath at Guy’s & St. Thomas’s Teaching Hospitals in London. Current research interests are around applications of long sequencing read technology in genetic diagnosis.
Nanopore sequencing in veterinary virology: from clinical sample to whole genome
avipoxviruses (APV), resulting in cutaneous and/or tracheal lesions, and belong to the Poxviridae family as well as the variola virus. Poxviruses share large genome sizes (from 130 to 360 kb), featuring repetitions, deletions or insertions as a result of a long-term recombination history. This disease may have a major economic impact in gallinaceous poultry and is an emerging concern for wildlife. Two independent cases of fowlpox were diagnosed in commercial layer farms in western France. All tracheal swabs and tissues sampled in both farms tested PCR positive for fowlpoxvirus. Using Oxford Nanopore Technologies sequencing, we readily generated whole APV genomes from cutaneous or tracheal lesions, without any isolation or PCR-based enrichment. Fowlpox virus read loads ranged from 0.75% to 2.62 %. The long read size eases the assembly step and lowers the bioinformatics capacity requirements and processing time compared to huge sets of short reads.
Guillaume completed a PhD in 2017 on the detection and characterization of respiratory pathogens using high-throughput PCR and next-generation sequencing methods. In November 2017 he joined the Chair of Biosecurity at the Veterinary School in Toulouse as an engineer in charge of the Clinical Diagnostics lab and development of new molecular tools for the detection and characterization of viral genomes.
Point of care Mycobacterium tuberculosis whole genome sequencing in remote rural Madagascar
Tuberculosis (TB) remains the leading infectious disease killer globally, with 10.4 million new cases in 2016 among which 4.1 million remain undiagnosed. Current approaches to TB control are predicted to fail to meet the World Health Organization objective to eliminate TB by 2030. With the rise of the multi-drug resistant tuberculosis (MDR-TB) epidemic, new innovative TB case finding, diagnosis, and control strategies are needed. Our objective is to achieve real-time TB diagnosis at the point of care, comprehensive genotypic drug susceptibility testing, and molecular epidemiology driven interventions in low resource, high burden settings. In April 2018, the Pasteur Institute of Madagascar, University of Oxford’s Modernizing Medical Microbiology group and Stony Brook University’s Global Health Institute launched a prospective pilot project which includes methods development for TB sequencing from sputum, and integration of portable TB DNA sequencing within National TB Program (NTP) clinical infrastructures and in collaboration with clinicians and policymakers.
Simon Grandjean Lapierre is a trained infectious diseases specialist and medical microbiology physician with previous clinical, laboratory and infection control experience in sub-Saharan African countries. He holds post-doctoral graduate degrees in global health and molecular diagnostics applied mycobacterial diseases. He currently works as a clinical lecturer in Centre Hospitalier de l'Université de Montréal in Canada, and coordinates the tuberculosis research program of Stony Brook University in Madagascar. His research activities focus on the implementation of new point of care and highly fieldable technologies for tuberculosis control.
Niaina Rakotosamimanana is a microbiologist and lab director of the Mycobacterial Unit of the Pasteur Institute of Madagascar in charge of the tuberculosis research program. This includes operational research that evaluates new TB diagnostic tools and drug resistance surveys in collaboration with the National Tuberculosis Control Program (NTCP) of the Malagasy Ministry of Public Health. He is in the steering committee of the international network for data analysis that aims to coordinate NGS data handling and NGS capacity building inside the Pasteur Institute international network that is present in 33 countries worldwide.
PromethION sequencing of complex plant genomes
KeyGene operates at the forefront with respect to new technologies and innovations in the field of plant genomics, and participates in the PromethION Early Access Program (PEAP). Genome sequencing initiatives of large, complex genomes typically yield highly fragmented genome assemblies. Using the PromethION, that offers ultra-long reads and high sequence output, KeyGene aims to produce contiguous, high-quality genome assemblies of plant pathogens and complex plant genomes. Recently we finished the data generation of the 2.7 Gbp lettuce genome and generated >100X coverage with just a few flow cells. To obtain high quality libraries for sequencing, the HMW DNA quality (integrity and purity) is crucial. KeyGene has developed specific knowledge in this area, and its impact on read length and yield will be presented and discussed.
Alexander Wittenberg graduated with an MSc in plant breeding and crop protection at the Wageningen University and completed his PhD at the Laboratory of Plant Breeding. Here he focused on the development of innovative genotyping methods to study the origin of genome plasticity in crop plants and their wild relatives. In 2007 he joined KeyGene, where he continued his work on the development and application of molecular marker methods. Alexander acquired considerable experience in the field of next-generation sequencing, with expertise on a wide range of platforms and applications. Currently he is a scientist contributing to the development of sequence-based technologies in KeyGene’s accelerated molecular breeding platform. Alexander was involved in the early evaluation of the technology during the MAP, and is now actively involved in the PromethION early access program, GridION and VolTRAX (VIP), as well as bringing this technology to the market for KeyGene’s clients.
Datema, E. et al. The megabase-sized fungal genome of Rhizoctonia solani assembled from nanopore reads only. bioRxiv 084772 (2016). doi:10.1101/084772
PromethION: an alpha machine in production
Andreas Hauser is a Staff Scientist, for the Laboratory for Functional Genome Analysis, at Gene Center Munich, LMU. He is also an entrepreneur.
Remmert, M., Biegert, A., Hauser, A. & Söding, J. HHblits: Lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nature Methods 9, 173–175 (2012).
Greif, P. A. et al. Somatic mutations in acute promyelocytic leukemia (APL) identified by exome sequencing. Leukemia 25, 1519–1522 (2011) doi: 10.1038/leu.2011.114.
Rapid bacterial identification from clinical samples using nanopore sequencing
Rapid detection of the pathogen is the most important step in the management of patients with infectious diseases. In this talk, I will share my research experiences of rapid bacterial identification from clinical samples using nanopore sequencing. Sequence-based pathogen identification directly from clinical samples has many advantages over the traditional culture-based methods. Nanopore sequencing of the 16S rRNA gene enables rapid diagnosis of bacterial infections, including polymicrobial infection and anaerobic infection, which will be very useful in the real clinical setting.
Jangsup Moon, MD, PhD is a neurologist interested in metagenomic pathogen detection using nanopore sequencing. He received his MD in 2005 and finished his Neurology residency training at Seoul National University Hospital in 2013. He received his PhD in Neuroscience at the College of Medicine, Seoul National University School in 2015. He is currently working as an Assistant Professor in the Department of Neurosurgery and Neurology at Seoul National University Hospital.
Moon, J. et al. Campylobacter fetus meningitis confirmed by a 16S rRNA gene analysis using the MinION nanopore sequencer, South Korea, 2016. Emerging Microbes and Infections 6,(2017)
Resistome accumulation induced by discharge of treated domestic wastewater - an overlooked aspect of modern wastewater treatment
Nanopore has become the leading 3rd generation sequencing platform in recent years, and has been widely applied in clinical and environmental researches with promising outcomes. In the present study, nanopore metagenomic sequencing was applied to investigate the microbiomes and antibiotic resistance genes (ARGs) from wastewater treatment plants (WWTPs) and the environment impacted by the WWTPs effluent, which include 1) resistome quantification in influent, activated sludge and effluent of WWTPs, 2) pathogen regrowth in the receiving water after treatment, 3) phenotype-genotype correlation (including both chromosome and plasmid) of multi-drug resistant coliforms in treated effluent and pure culture of E. coli. A convenient and efficient workflow was developed for rapid ARGs detection and host tracking based on nanopore data sets. Overall, this work presents rapid and efficient resistome characterization by nanopore technology that could facilitate ARGs monitoring and control in hotspots, such as WWTPs and the impacted environments.
Yu Xia is Assistant Professor of Southern University of Science and Technology (SUSTech), and received her PhD from the University of Hong Kong. Her research interest focuses on utilizing nanopore sequencing to understand the community ecology and functional synergy among microbes that are crucial for biological wastewater treatment or important for nature material cycles. The topics she currently works on include: environmental dissemination of antibiotic resistance genes (ARGs); direct interspecies electron transfer (DIET) capable microbes in anaerobic sludge digestion; sulfate/nitrate reduction mediated anaerobic methane oxidation (AOM) in coastal sediment. Additionally, she has served as the Young Ambassador of Hong Kong region for American Society for Microbiology (ASM) and the General Secretary for the Postgraduate Student Association (PGSA) of the University of Hong Kong.
Simultaneous methylation and chromatin accessibility profiling on breast cancer models
The ability of nanopore sequencing to distinguish modified nucleotides has tremendous potential in exogenous labeling, where one can methylate DNA in a given state, e.g. nucleosome positioning or interactions with specific proteins. Once the DNA is exogenously methylated, we can take advantage of the long-read lengths (>10kb) generated by nanopore sequencing to call methylation across stretches of genomic regions on individual reads. We have previously shown that endogenous CpG methylation can be accurately called using nanopolish, and we have expanded nanopolish modification detection pipeline to detect GpC methylation. We used these models to simultaneously profile nucleosomes and methylation directly on long-reads. Aberrant gene regulation is the source of many diseases including cancer. Two key effectors of this are genomic instability and chromatin states. NOMe-seq has been used previously to characterize chromatin state by probing the endogenous CpG methylation and exogenously labeled nucleosome occupancy. By exogenously labelling nuclei with GpC methyltransferase, cytosines in GpC dinucleotides are methylated in nucleosome-depleted, accessible genomic sites. We have successfully applied this methodology to nanopore sequencing, using nanopolish modification detection pipeline to detect CpG and GpC methylation. We show that using nanoNOMe we can precisely detect nucleosome occupancy along with methylation on individual long-reads. Using the long-reads, we can observe haplotypes, patterns, and correlations of the modifications. Applying nanoNOMe to study the epigenetic state of cancer, we demonstrate our results from performing deeply sequenced nanoNOMe on breast cancer cell lines exhibiting various degrees of malignancy, including MCF10A, MCF-7 and MDA-MB-231.
Isac Lee is a PhD student in the department of Biomedical Engineering at Johns Hopkins School of Medicine. He is being trained in the lab of Dr. Winston Timp, studying the epigenome and genome of cancer. He is interested in epigenomic reprogramming and its association with gene expression and genomic aberrations during cancer progression. He is working on integrating and developing genomic and epigenomic methodologies, and applying them on cancer models and clinical samples.
The complex architecture of plant T-DNA transgene insertions
Over the last 35 years the soil bacterium Agrobacterium tumefaciens has been used as the workhorse in the production of transgenic plants through the replacement of its native tumor-inducing plasmid elements with customizable cassettes that enable the random integration of a Transfer DNA (T-DNA) sequence into any plant genome. Due to previous limitations in sequencing read lengths, the architecture of these T-DNA insertions has gone largely uncharacterized. We leveraged Oxford Nanopore Technologies long-reads in combination with optical maps to characterize T-DNA insertions, ranging from 27 to 236 kilobases in the model plant Arabidopsis thaliana. We generated a higher quality reference assembly, which corrected 83% of non-centromeric misassemblies in the platinum hand-curated TAIR10 assembly. For two segregating T-DNA lines we resolved structures up to 36 kb and revealed large-scale translocations events. This unprecedented nucleotide-level definition of T-DNA insertions enabled the characterization of the epigenomic status associated with the insertions.
Dr. Todd Michael is Professor and Director of Informatics at the J. Craig Venter Institute (JCVI) in San Diego, CA USA. At JCVI, Dr. Michael’s group builds genome sequencing, editing and analysis tools with a specific focus on developing synthetic plants. Current projects include sequencing minimal and specialized plant genomes, computationally designing a minimal plant genome, booting-up plant artificial chromosomes, and leveraging nanopore sequencing for complete genomes and epigenomic analysis.
Jupe, F. et al. The complex architecture of plant transgene insertions. bioRxiv 282772 (2018). doi:10.1101/282772
Michael, T. P. et al. High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell. Nature Communications 9, (2018).
Understanding transmission dynamics of TB and AMR in the farmland environment
Environmental reservoirs of infection within the agricultural landscape are of particular concern because of the risk of indirect transmission between livestock and humans. Long-read sequence technology provides a valuable tool to help better understand dissemination from the environment due to the complexity of environmental matrices, such as soil and faeces. We firstly present a novel method to strain type Mycobacterium bovis, the causative agent of bovine tuberculosis, from both environmental matrices and clinical isolates within the UK. Given the genetic homogeneity of the mycobacterium complex, long-read sequencing was used to genotype M. bovis by quantifying six VNTR regions and the direct repeat region. This has permitted us to develop a rapid and responsive strain typing tool for M. bovis that circumvents the requirement for culture. Furthermore, we present a novel use of the Oxford Nanopore sequencing platform to investigate the drivers and dissemination of antimicrobial resistance in the environment within, and between, livestock producing and residential areas of Karachi, Pakistan. Amplification and sequencing of Class I intergrons, a mobile genetic element often associated with antimicrobial resistance, was used as a proxy for the environmental resistome. Results from this pilot study indicate that the environmental resistome of the two areas were significantly different, with a greater dissimilarity in AMR abundance and diversity seen within chromosomally incorporated integrons.
Dr Robert 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, Robert 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, Pakistan.
VirION: Towards long-read viral metagenomics