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.

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.

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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.

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.

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.

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.

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.

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.

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.

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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.

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.

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.

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.

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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.

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.

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).

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.

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.

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.

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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.

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.

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.

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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.

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.

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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.

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.

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.

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.

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Viruses are the most abundant organisms in the ocean and grease the wheels of global carbon biogeochemistry. Recently, our understanding of viral ecology has taken a major leap forward with the use of viral metagenomics. However, short-read sequencing used for this purpose has its drawbacks — many of the most abundant and important viral taxa fail to assemble and are thus lost from our studies. Here, I will present new methods for using the MinION for long-read viral metagenomics. I will show that these new methods dramatically increase viral diversity captured in marine samples, and provide insight into host-virus interactions in a way that cannot be achieved with short-read technology. I will share our bioinformatic pipelines to improve sequence fidelity, and discuss how MinION technology is bringing us closer to high-throughput single-virus genomics from environmental samples.