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.
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.
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.
The phyllosphere of Euphorbia lateriflora and Ficus thonningii
The phyllosphere, which includes the microbiome of the leaves of plants, remains an understudied ecological niche. The bacterial colonizers of medicinal plants have barely been studied even though their host plants have been used widely in ethnomedicine. Part of the limitations of studying phyllosphere bacteria is that they cannot be sufficiently classified using traditional culture and biochemical methods, largely because such methods were created with a focus on medically important bacteria which represent only a small fraction of the group. Consequently, phyllosphere studies conducted before the era of genomics were mostly species in-specific or focused on bacterial pathogens/symbionts of food plants. It soon became clear as phyllosphere studies progressed that some organisms, including novel ones, were missed as they were not covered even in the classic Bergey’s manual of systemic bacteriology database. With the advent of sequencing, it has become possible to study phyllosphere bacteria in detail, both culturable and unculturable. Metagenomic DNA can be obtained directly from plant colonizers and sequenced directly. Phyllosphere studies currently employ sequencing to characterize bacteria, including novel species. We used sequencing to characterize phyllosphere bacteria of Ficus thonningii and Euphorbia laterifolia which are Nigerian medicinal plants, hypothesizing that both plants are colonized by diverse bacteria which are advantageous. Bacteria were isolated on 0.1X tryptone soya agar, after which isolates were identified by 16S rRNA gene sequencing, compared by multiple sequence alignments and phylogenetics. Select isolates were further classified biochemically with Microbact 24E and genotypically by whole genome sequencing. A total of 100 and 77 isolates from F. thonningii and E. lateriflora respectively were identified by 16S rRNA sequencing. The dominant phylum and family from both plants were Proteobacteria and Enterobacteriacea respectively. 12 other bacterial families were encountered. Biochemical and genomic classification of 14 isolates which could not be assigned to any species based on 16S rRNA analyses were discordant as Microbact identified all but two as Acinetobacter, while genome sequencing classified them as Brevibacteria, Agrococci, Kocuria and others. Plasmids, resistance and virulence genes were also detected in a sub set of isolates. Study of phyllosphere bacteria was greatly limited by traditional microbiology methods up until introduction of sequencing into the field. Apart from correctly identifying organisms, sequencing provides extensive insight into phylogeny and metabolic capacity of organisms, making it possible to predict functions of the bacteria to host plants after a single sequencing experiment. This has greatly changed the ways in which the phyllosphere microbiome is studied.
Anderson O. Oaikhena is a Doctoral student and teaching assistant in the Department of Pharmaceutical Microbiology at the University of Ibadan. He is currently studying bacterial colonizers of medicinal plants, with a particular interest in colonization of antibacterial plants as a framework for better understanding the ecology of antimicrobial resistance in nature. Anderson’s outstanding performance in the Master of Science degree in Pharmaceutical Microbiology in 2017 earned him a doctoral scholarship award from the postgraduate college of University of Ibadan. He serves as a research associate in the Nigeria AMR surveillance network, where he is the point person for whole genome sequencing of antibiotic resistant bacteria.
Genomic profiling in acute myeloid leukemia with complex karyotype
Acute myeloid leukemia (AML) represents clonal expansion of malignant cells. A stratification of patients in risk groups is based on cytogenetics and molecular markers for a genotype-based treatment strategy. Conventional karyotyping, which is necessary for classification of “high-risk” AML, is available after 5 to 7 days. Using Oxford Nanopore sequencing, we established karyotyping based on shallow genome sequencing within 24 hours. The throughput of one flowcell was sufficient to achieve 3-fold genome coverage and reproduce results of conventional karyotyping in 20 AML patients. To discover structural variations, we applied direct RNA sequencing and analysed fusion genes based on 1.2 million reads. A single run is sufficient to detect a balanced translocation t(9;22), a fusion gene BCR-ABL1, in the cell line K-562. While a study of a larger AML patient cohort is ongoing, parallel low coverage genome and transcriptome analysis allows identification of high-risk AML during the initial diagnostic work-up of 24 hours.
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.
MinION sequencing from the extreme to the everyday
Dr. Arwyn Edwards is Senior Lecturer in Biology and Director of the Interdisciplinary Centre for Environmental Microbiology at Aberystwyth University. Portable DNA sequencing using the MinION is enabling Arwyn and other Arctic microbiologists respond to rapid changes in the Arctic, and access unprecedented insights into the diversity and function of extreme microbial ecosystems. This area of research is supported by NERC, Leverhulme, the Welsh Government and the European Union.
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.
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.
The Ohio State University
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.
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.
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.
Redefining the transcriptional complexity of viral pathogens using direct RNA sequencing
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
Short-read sequencing platforms have been adopted by public health agencies for infectious disease surveillance worldwide and have proved to be a robust and accurate method for quantifying relatedness between bacterial genomes. However, this approach offers less flexibility for urgent, small scale sequencing that is often required during public health emergencies. In contrast, Oxford Nanopore Technologies offers a range of rapid real-time sequencing platforms, although at this time it has been suggested that lower read accuracy compared to other sequencing technologies might be problematic for variant identification. We compared Illumina and Oxford Nanopore sequencing data of two isolates of Shiga toxin producing Escherichia coli to assess the utility of nanopore technologies for urgent, small scale sequencing. We investigated whether the same single nucleotide variants were identified by the two sequencing technologies and whether inference of relatedness was consistent. We show that with optimised variant calling using nanopore sequencing data alone, it is possible to rapidly determine whether or not two cases of were likely to be epidemiologically linked.
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.
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.
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.
Center for Genomic Regulation
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.
Genomics from roadkill: high quality mammalian genomes using hybrid assembly with MinION long reads
Frédéric Delsuc is Research Director at the French National Centre for Scientific Research (CNRS), working in the Institute of Evolutionary Sciences at the University of Montpellier. He received his PhD in molecular phylogeny from the University of Montpellier, then worked on mammalian and tunicate phylogenomics during post-doctoral positions in New Zealand and Canada before coming back to Montpellier as a permanent CNRS researcher. He is currently directing the ERC ConvergeAnt project aimed at understanding convergent evolution in ant-eating mammals through an integrative approach combining morphology, genomes, and microbiomes. The project team has adopted nanopore sequencing technology using the MinION to produce long-reads combined with Illumina short-reads to assemble mammalian genomes mostly from roadkill animals.
University of Oxford
George and his colleagues from the University of Oxford Big Data Institute have recently returned from the Mobile Malaria Project, a six-week trip to Africa to learn about malaria research and to trial MinION sequencing pipelines in the field. Working with local collaborators in Zambia and Kenya, much of the project was spent training African scientists on the basics of nanopore sequencing and assessing its feasibility in low resource settings. The team tested amplicon sequencing pipelines for key antimalarial resistance genes in Plasmodium falciparum and insecticide resistance genes in Anopheles gambiae. They hope to use what they learnt to further develop research collaborations and build capacity for nanopore sequencing in Africa.
Long reads reveal small scale genome structural variations in Brassica napus
In this era of climate change and global warming it is our responsibility as the scientific community to find sustainable ways for meeting our energy and fuel requirements. Canola, Brassica napus, based biodiesel provides a perfect alternative to the use of fossil fuels and can help us cut our Greenhouse gas emissions by up to 90%. In order to counteract the ever-increasing demand for fuel and energy, it is crucial to maintain a high yield for this crop without generating a huge environmental footprint. However, B. napus is a very complex genome originating from an inter-specific hybridization event between Brassica oleracea (Mediterranean cabbage) and Brassica rapa (Asian cabbage or turnip). Due to high levels of homology between the two sub-genomes, making up the Canola genome it is extremely difficult to identify the novel genome polymorphism underlining important traits such as yield, disease resistance and abiotic stress tolerance. Next generation genome sequencing had been a game changer when it comes to deciphering complex quantitatively inherited traits in B. napus. However, the resolution offered by the second-generation sequencing technologies, such as Illumina sequencing, was severely limited due to the small size of the sequencing reads. With Oxford Nanopore technology it is now possible to zoom into the Canola genome to identify gene level structural variation associated with key traits such as yield. We have sequenced 4 Canola genotypes using nanopore technology and identified insertions and deletions ranging from 50bp to 10,000bp in genes involved in a plethora of important traits like disease resistance, flowering time etc. This knowledge will enable us to engineer a future ready Canola plant.
Harmeet Singh Chawla is a PhD student in the Department of Plant Breeding at the Justus Liebig University Giessen. Harmeet completed a MSc in Agro-biotechnology at JLU Giessen, and is interested in studying the impact of genome structural variations on eco-geographical adaptation and various other agronomically important traits in Brassica napus, Canola.
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.
Small, modified and highly structured: the challenge of tRNA sequencing
Oxford Nanopore Technologies have been initially developed to directly sequence the long molecules of DNA and RNA. The possibility of sequencing shorter molecules using nanopore is widely discussed in the field but remains questionable because of the higher error rate compared to the classical deep-sequencing approaches. Here we show the successful application of the MinION device to sequence tRNA molecules. The challenges of sequencing tRNAs are due its short length and folded structure, however, to overcome this we have improved the library preparation in order to compute the whole length of the tRNA. Initially, we sequenced a mixture of in vitro transcribed E. coli tRNAs and developed a bioinformatical pipeline to assign base-called reads to different tRNA species with a high degree of accuracy. Moving towards more complex samples containing native tRNAs, we found that modifications along the tRNA reduced the fidelity of called bases, so we developed an algorithm of tRNA classification based on raw-signal patterns. Comparing those patterns to unmodified in vitro transcribed tRNA signals allowed us not only to distinguish between different tRNA species, but also to detect modifications occurring in the native tRNAs. Our results show that nanopore-based approaches can be used to sequence tRNAs and classify them. This unveils a new area of the nanopore technology in application to short molecules, detecting the modifications and even predicting the potential ones, which are currently unknown, but may govern the structure, affect decoding or play a role in diseases.
Irina studied Medical Cybernetics at the Russian National Research Medical University in Moscow and graduated with a Medical Degree. She then completed a Masters in Biochemistry and Bioinformatics at the University of Potsdam in 2015, where she began working with NGS data. Irina is currently a PhD student at the Institute of Biochemistry and Molecular Biology at the University of Hamburg. As a bioinformatician, Irina processes various deep sequencing data including nanopore and develops new algorithms for analysis.
SquiggleKit: a toolkit for manipulating nanopore signal data
The management of raw nanopore sequencing data poses a challenge that must be overcome to accelerate the development of new bioinformatics algorithms predicated on signal analysis. SquiggleKit is a toolkit for manipulating and interrogating nanopore data that simplifies file handling, data extraction, visualisation, and signal processing. Its modular tools can be used to reduce file numbers and memory footprint, identify poly-A tails, target barcodes, adapters, and find nucleotide sequence motifs in raw nanopore signal, amongst other applications. SquiggleKit serves as a bioinformatics portal into signal space, for novice and experienced users alike. It is comprehensively documented, simple to use, cross-platform compatible and freely available (https://github.com/Psy-Fer/SquiggleKit).
James Ferguson is a Genomic Systems Analyst in the Genomic Technologies Group at the Kinghorn Centre for Clinical Genomics, located at the Garvan institute of Medical Research in Sydney, Australia. With a background in clinical pathology testing, algorithm development, and computer hacking, James applies his unique skill set to develop new bioinformatic tools, as well as design and support nanopore sequencing infrastructure.
Mobile Malaria project
Cyclomics: ultra-sensitive nanopore sequencing of cell free tumor DNA
Dr. Jeroen de Ridder is a Principal Investigator and Associate Professor at the Center for Molecular Medicine of the University Medical Center Utrecht, as well as a junior PI at the Oncode Institute. He runs a bioinformatics lab which aims to create and apply innovative data science methods to advance our understanding of disease biology. His research efforts are always inspired by a biological question and typically deal with big data, such as large-scale genomics and epigenomics datasets. As a result, much of the research floats on machine learning and data integration algorithms. Recently, Dr. de Ridder, along with Dr. Kloosterman and Dr. Marcozzi, founded a start-up company Cyclomics, which aims to provide ultra-sensitive sequencing of cell free tumor DNA.
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.
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.
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.
Single cell isoform profiling, 10xGenomics scRNA-seq and nanopore long read sequencing
Single cell transcriptome sequencing has become a powerful tool for high-resolution analysis of gene expression in individual cells. However, current high throughput approaches only allow sequencing of one extremity of the transcript (transcriptome profiling). Information crucial for an in-depth understanding of cell-to-cell heterogeneity on splicing, chimeric transcripts and sequence diversity (SNPs, RNA editing, imprinting) is lost. Here we present an approach that uses Oxford Nanopore sequencing with unique molecular identifiers to obtain error corrected full length single cell sequence information with the 10xGenomics single cell isolation system and apply it to examine differential RNA alternative splicing and RNA editing events in the embryonic mouse brain.
Kevin Lebrigand is Head of Bioinformatics at UCAGenomiX, the functional genomics platform of Nice-Sophia-Antipolis, one of the core nodes of the "France Genomique" network, using next generation sequencing to perform a broad range of sequencing projects such as de novo genome assembly, RNA-seq, small RNA-seq, CHIP-seq and CLIP-seq. In 2014 the platform decided to focus their expertise on methodological developments around single cell transcriptomics using the Fluidigm C1, and more recently the 10xGenomics Chromium system, on which more than 120 samples has been profiled. Last summer Kevin acquired a PromethION long-read sequencer to perform isoform-level profiling at the single cell level.
Retrotransposon variation in human genome and tumorigenesis
Retrotransposons are transposable genetic sequences that copy themselves into an RNA intermediate and insert elsewhere in the genome by reverse transcription. Almost half of the human genome is derived from transposon derived sequences but only some dozens of full length Long Interspersed Nuclear Element-1s (LINE1s) in the human germline are expected to be retrotransposition competent. Mapping and genotyping retrotransposons with short-reads is complicated due to their size and high copy number of their consensus sequence in the reference genome, so we applied nanopore sequencing to study multiple aspects of LINE1 retrotransposition. First, we sequenced Inverse-PCR products with MinION detecting highly subclonal insertion sites of a particular LINE1 element in two colon cancer tumors. Second, we have whole genome sequenced few germline and tumor genomes with PromethION, detecting the whole range of retrotransposon insertions that are variable within humans or inserted during tumorigenesis. Finally, we studied DNA methylation around the LINE1 insertion and source sites in human tumors in order to understand the mechanisms of LINE1 activation in during tumorigenesis. Presently we are extending these studies to ~300 whole genomes of Uterine Leiomyoma tumors and their respective normal sequences.
PSI-Sigma: a comprehensive splicing-detection method for short-read and long-read RNA-seq analysis
Percent Spliced-In (PSI) values are commonly used to report alternative pre-mRNA splicing (AS) changes. Previous PSI-detection tools were limited to specific AS events and were evaluated by in silico RNA-seq data. We developed PSI-Sigma, which uses a new PSI index, and we employed actual (non-simulated) RNA-seq data from spliced synthetic genes (RNA Sequins) to benchmark its performance (i.e., precision, recall, false positive rate, and correlation) in comparison with three leading tools (rMATS, SUPPA2, and Whippet). PSI-Sigma outperformed these tools, especially in the case of AS events with multiple alternative exons and intron-retention events. We also briefly evaluated its performance in long-read RNA-seq analysis, by sequencing a mixture of human RNAs and RNA Sequins with nanopore long-read sequencers. Based on the long-read RNA-seq data of RNA sequins, we found that nanopore long-read RNA-seq is qualitatively reliable. Also, in human U87 cells, we found that ~1 million long reads can already detect major AS changes in ~3,500 protein-coding genes with at least 10 supporting long reads. PSI-Sigma is implemented in Perl and is available at https://github.com/wososa/PSI-Sigma
Kuan-Ting Lin is a Computational post-doc at Cold Spring Harbour Laboratory, where he focusses on quantitative biology and transcriptomics technologies. His long-term research interests involve the use of mathematical, statistical or computational techniques to develop understanding of how alterations in RNA transcription contribute to human health and his academic training and research experience has provided an excellent background in drug discovery, data mining and quantitative biology.
Nano-C: targeted poly-contact chromatin interactions for comprehensive profiling of cell-to-cell variation of 3D genome organization
Li-Hsin Chang is a postdoctoral researcher at the Chromatin Dynamics group led by Dr. Daan Noordermeer at the Institute for Integrative Biology of the Cell (I2BC) in France. Li-Hsin received her PhD in cellular and developmental biology in 2017 from the University of Illinois at Urbana-Champaign, where she studied the function and regulatory landscape of zinc-finger transcription factors with Professor Lisa Stubbs. Her current research interests focus on 3D chromatin organization, aiming to uncover the cellular heterogeneity of Topologically Associating Domains (TADs). To this end, she has developed a new method “Nano-C”, which combines chromosome conformation capture (3C) technique with nanopore sequencing for detecting poly-contact chromatin interactions.
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.
After graduating from the Sam Ratulangi University Manado in Indonesia, Lucky Ronald Runtuwene worked as a medical doctor in a community health center on the island of Siau. There he became interested in infectious diseases, and so decided to pursue a research career in the field. He completed a PhD in Japan, where he probed the gene expressions of a vector mosquito infected with dengue virus followed by a post-doc in the University of Tokyo, where his laboratory was one of the early adopters of the MinION technology. Lucky is interested in field work and so the portability of the MinION and ease of processing aid his research, leading to the conception of the GRAID consortium which he will introduce at London Calling 2019.
Mapping DNA replication using nanopore sequencing
Magali Hennion completed a PhD in molecular biology in Toulouse with Dr Emmanuel Käs and Dr Olivier Cuvier, where she worked on chromatin organisation and insulator proteins. She then moved to Göttingen in Germany where she worked with Dr Steven Johnson in a postdoc internship on chromatin changes during stem cell differentiation, before a second postdoc with Stefan Bonn, where she focussed on epigenetic changes associated with memory formation and maintenance in the mouse brain. Since 2016, Magali has worked in Dr Olivier Hyrien's team in Paris, where she is developing new techniques to study DNA replication based on nanopore sequencing.
CSIR - National Chemical Laboratory
Using full-length transcript sequencing to reveal the fate of mRNA in aging seeds
After seeds mature on the mother plant, they contain all the molecular machinery they will need for germination. Generally, some time elapses between maturation and germination. At the National Center for Genetic Resource Preservation, this time may be months to decades, or, optimistically, centuries. But, during this time, seeds eventually lose the ability to germinate. One explanation for this change is that the molecular machinery has gradually accumulated damage. When trying to assess the health of a seed lot, the standard method is to use a subset of seeds for a germination test, but these results are not straightforward to interpret. We hypothesized that accumulated damage at the molecular level could be quantified as an independent measure of seed lot health and chose to examine RNA. We showed that integrity of total RNA declines with storage time in seeds of many species. To show whether mRNA was similarly affected, we compared transcript integrity in 23-year-old and 2-year-old soybean seeds by sequencing full-length cDNA using MinION. In 23-year-old seeds, certain transcripts were only partially sequenced, and we confirmed that this was because of transcript fragmentation at random sites. We quantified transcript degradation for all transcripts and found that degradation increased with transcript length. This result supports the hypothesis that random damage accumulates at the molecular level. We now anticipate using the integrity of long transcripts to assess seed health over time.
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).
Genome and Plant Development Laboratory
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.
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.
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.
University of Nottingham
Plant de-novo genome sequencing and assembly using Oxford Nanopore Technology
Oxford Nanopore sequencing technology has made it possible to sequence and de novo assemble plant genomes at relatively low cost with fast turn-around times. Continuous improvements in the technology as well as our optimization of DNA extraction, size selection and library preparation make it possible for us to assemble larger plant genomes to a high-quality draft level. Using Hi-C we are further able to improve those genomes to chromosome-scale assemblies.
Maximilian Schmidt was awarded a BSc in Biotechnology from the University of Cooperative Education Riesa in 2012, before moving to RWTH Aachen to complete a MSc, where he studied genes involved in plant cell wall biosynthesis. He is currently a PhD student with Prof. Usadel at RWTH Aachen where he is interested in de-novo plant genome sequencing.
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.
Deep transcriptomic sampling with long-read single cell RNA sequencing
Mike Clark is head of the Transcriptomics and Neurogenetics group at the University of Melbourne in Australia. His research sits at the intersection of genomics and neuroscience, utilizing a number of genomic approaches, including nanopore sequencing, to investigate gene expression and function in the human brain and in neuropsychiatric disorders.
It is all about accessibility: Galaxy as a framework for democratizing Oxford Nanopore data analysis
Thanks to Oxford Nanopore Technologies, long-read sequencing is becoming more accessible for a much broader range of applications and end-users. Bioinformatics analysis was already a bottle-neck with the previous generations of sequencing technologies, but even more so with the new generations. Nanopore-based sequencing technologies are so much more accessible and can rapidly produce so much more data that the data analysis challenges can become fundamental. Community-driven solutions to democratize data analysis is crucial in the same way Oxford Nanopore is democratizing sequencing. Galaxy has been shown to be a successful option for short-read sequencing, but we think its advantages will shine even more in the era of long-read sequencing. Firstly, the user-friendly web interface does not require advanced computational skills, making it ideally suited for this interdisciplinary area and educational purposes. Secondly, the software and workflows can be seamlessly upgraded at the server side while maintaining 100% reproducibility of the performed analysis. Thirdly, the computational infrastructure supports a diverse spectrum from personal computers to cluster grids and the cloud. Within the scope of this project, we provide Oxford Nanopore-related tools in Galaxy. We have developed a collection of the best practice workflows for genome assembly within Galaxy. Our work is available for everyone at the European Galaxy server (https://usegalaxy.eu) and supportive self-learning training material is available. I will also introduce the Street Science Community (https://streetscience.community), a voluntary-based non-profit group that aims to teach the public the fundamental concepts of molecular biology and genetics data analysis by analyzing the “DNA of beer” using MinION and Galaxy.
Milad Miladi is PhD candidate and research assistant in the Bioinformatics group at the University of Freiburg in Germany. With a background in computer science and RNA computational biology, his research involves transcriptomics, non-coding RNAs and reproducible data analysis with Galaxy.
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.
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 modifications, and software for analysis of MinION and PromethION data.
Characterizing large homology directed repair (HDR) insertions by CRISPR/Cas9 using MinION long-read sequencing technology
Mollie Schubert is a Research Scientist in the molecular genetics research group at Integrated DNA Technologies. Mollie received her master’s degree in biochemistry from Iowa State University and has been at IDT since 2013. For the past five years, she has focused on studying CRISPR gene editing, including high-throughput screening of CRISPR-Cas9 guides for the development of a site selection tool, optimizing the composition and delivery of synthetic RNA reagents complexed to recombinant CRISPR nucleases, and developing methods for efficient gene editing with a recent focus on improvements to homology directed repair.
Department of Primary Industries and Regional Development
Resolution of germline hereditary cancer structural variants using nanopore sequencing
Dr. My Linh Thibodeau is currently training in the Medical Genetics Residency Program at the University of British Columbia. In 2017, she won entry to the Royal College of Physicians and Surgeons Canada Clinician Investigator Program to apply bioinformatic approaches to the discovery and characterization of hereditary cancer predispositions. During her work in the Personalized OncoGenomics study at BC Cancer in Vancouver, Canada, Dr. Thibodeau acquired expertise in the analysis and integration of whole genome and whole transcriptome datasets. Taken together with her medical training, these experiences have allowed Dr. Thibodeau to develop a unique clinical-bioinformatic skillset.
University of Nottingham
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.
Blood donor genotyping - how can long range sequencing help?
Nicholas Gleadall is a PhD student working in the laboratory of Professor Willem Ouwehand at the University of Cambridge. His work focuses on the genetics of human blood group antigens and development of techniques for high throughput, DNA based donor typing. Nicholas has previous experience introducing new technologies into clinical service by developing and validating diagnostic laboratory assays for large organisations such as Public Health England, where he worked on HIV whole genome sequencing for national surveillance and resistance genotyping, and NHS England on a project focussed on exome sequencing for diagnosis of rare human inherited disorders.
University of Birmingham
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.
Nanopype: processing and quantification of short tandem repeats
The availability of substantially longer reads with the Oxford Nanopore approach opens new possibilities in many fields and explains the increasing use of the nanopore technology. To facilitate access and match storage as well as processing routines to the higher demand, we assembled Nanopype a modular, parallelized and easy-to-use pipeline to process the sequencing data from the raw signal output into standardized formats. Specifically, Nanopype facilitates the essential steps of base calling, quality control, and alignments, as well as various downstream applications by incorporating field-specific tools and complemented by custom utility scripts. To illustrate its application, we apply it to the assessment of short tandem repeats that have been implicated in neuropsychiatric disorders. Combined with a Cas12a-based enrichment strategy and the STRique package we show efficient targeting and quantification on raw signal level, as well as determination of the associated methylation status.
Pay received his MSc in Electrical Engineering from the Kiel University of Applied Sciences with a focus on embedded systems and hardware accelerated signal processing. He is currently a PhD student in Alex Meissner's lab at the Max-Planck-Institute for Molecular Genetics in Berlin. Pay is interested in the epigenetic regulation of the genome, direct base modification detection and developing tools and pipelines to process third generation sequencing data.
University of Nottingham
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.
Biological evidence of the future: the use of sequencing in forensic DNA analysis
Forensic DNA profiling uses short tandem repeat (STR) analysis for human identification purposes, i.e. to establish a link between biological evidence and an individual. This technique is currently limited to assessing the length of STR alleles via capillary electrophoresis and relies on the comparison to a reference DNA profile. The advent of DNA sequencing has revolutionised the field of forensic genetics. Alleles with the same length but a different sequence can be distinguished, providing additional discrimination between individuals which can greatly aid in DNA mixture interpretation. Rare sequence mutations can be identified to differentiate identical twins, who cannot be told apart using conventional DNA profiling. Using sequencing, scientists have also begun to harness intelligence-based information that a biological sample can provide which could be of use in an investigation. The analysis of single nucleotide polymorphisms (SNPs) offers new opportunities in the form of forensic DNA phenotyping and forensic epigenetics. Prediction of eye, hair and skin colour, as well as bio-geographic ancestry and chronological age estimations of an unknown individual are all now possible. The introduction of nanopore sequencing technology has the potential to transform the field of forensic genetics even further. The portability and real-time capability of the MinION could shift analysis out of the lab into the field, greatly reducing cost and turnaround time which are critical in an investigation. Research into the feasibility of this technology for forensic applications is currently underway. Sequencing has not only changed the field of forensic genetics, but also has changed the way biological evidence is approached and could be used in investigations which has had a wide-reaching effect in enforcement, legal, governmental and judicial fields. Although not routinely used in forensic casework at present, many forensic laboratories around the world are currently validating sequencing technologies with the expectation that this will be the biological evidence of the future.
Rebecca Richards is a doctoral student in the Forensic Science Programme at the University of Auckland. Her research focuses on the development and optimisation of DNA methylation markers for forensic applications, specifically identical twin differentiation and chronological age estimation. Rebecca is also a senior technician in the Forensic Biology Group at the Institute of Environmental Science and Research (ESR), a Crown Research Institute which provides forensic services to the New Zealand Police. In addition, she is running point for the MinION research currently being undertaken at ESR and is involved in the wider validation of DNA sequencing for forensic use.
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.
Nanopore sequencing in space: one small step for a MinION, one giant leap for spaceflight research
As NASA sets sights beyond low-Earth orbit, real-time monitoring and diagnostics of crew health and the environment are required. The majority of previous research on the International Space Station (ISS) has relied on ground-based analyses following sample return to Earth. As a result, biological insights could be lost due to sample fixation and time to receipt in the laboratory. Moreover, sample return will become impractical as missions move beyond the ISS. Oxford Nanopore Technologies’ MinION has made in situ sequencing a reality in any field setting. Since our 2016 demonstration of the MinION’s high-performance off-Earth, four astronauts across the last ten ISS expeditions have successfully completed 18 sequencing experiments. Here, we will describe our end-to-end, sample-to-sequencer process that can be conducted entirely aboard the ISS, and which resulted in the first identification of microbes collected and cultured entirely off-Earth. Expanding beyond the need to first culture the microbes, our culture-independent, swab-to-sequencer investigation is currently underway to characterize further the microbiome of the ISS. One of our long-term goals is to enable crew health monitoring using functional genomics. As a start, we sequenced native poly-A RNA (and cDNA) from a human cell line (GM12878) aboard the ISS and will present findings from these experiments. Nanopore sequencing technology and our sample preparation procedures have expanded the reach of molecular biology to the final frontier. These proven capabilities hold the potential to revolutionize space-based research and in-flight medical operations.
Dr. Sarah Wallace serves as technical lead in the Microbiology Laboratory at the NASA Johnson Space Center, which is responsible for mitigating infectious disease risk during human spaceflight. Her responsibilities include the assessment of microbial risk based on vehicle and mission architectures as well as crewmember, food, and environmental monitoring. These assessments are used to develop requirements for NASA and commercial spaceflight vehicles, including the International Space Station. In addition to her operational support of human spaceflight, Dr. Wallace leads new technology initiatives for her discipline with the goal of reducing Earth dependence for complex sample analysis. She has served as PI for numerous spaceflight investigations, including those to increase off-planet molecular biology capabilities and also to understand how the spaceflight environment impacts cellular behavior.
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.
Unraveling shark secrets: sequencing genomes and microbiomes for research and conservation!
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.
Shaili Johri is a geneticist with a research focus in conservation genomics of wildlife populations. She completed her BSc. and M.S. in India and moved to the United States for her PhD in Genetics. She did her post-doctorate at the Center for Conservation Biology at the University of Washington in Seattle and is currently a research professor at San Diego State University in association with Dr. Elizabeth Dinsdale’s laboratory. Shaili works at the intersection of marine conservation policy and interdisciplinary research and her research career spans projects relating to conservation of tigers in western India, wolves in northwest USA, killer whales in the Pacific Northwest and now sharks and rays in the southwest US and India. Shaili’s role as a project lead involves developing genomic and metagenomic tools to assist with biodiversity assessments and population health monitoring of marine megafauna such as sharks and killer whales. In parallel to her research, Shaili works in close collaboration with fishing communities to develop science-based conservation policies through cross-sector collaborations, outreach and education. In addition to her research, Shaili engages in capacity building for genomic methods among wildlife research communities in the US and abroad and this is where the Oxford Nanopore MinION device has been a game changer.
Obtaining high quality DNA from plant tissues for nanopore sequencing
Stella is currently a platform coordinator at a small, not-for-profit sequencing centre and NGS training facility at Deakin Genomics Centre in Melbourne, Australia. She has been technical specialist at Deakin University for over a decade, providing training and support to undergraduate and post graduate students in a broad range of molecular and cell biology techniques. Stella completed her undergraduate degree in Science at Latrobe University and PhD at the University of Sydney, Australia. She has extensive experience working with difficult specimens such as formalin-fixed museum specimens, plants, insects and soil.
Applied bioinformatics: from basic QC to Epi2ME
Stephen Rudd joined the Product management team last year having previously been the Strategic Account Manager at Oxford Nanopore Technologies for customers in Germany and Austria. Stephen is a classical geneticist and has a background in genome bioinformatics. He has been project manager for a taxonomically diverse range of genome studies utilising most DNA sequencing and genotyping technologies. He is looking forward to brainstorming potential solutions to challenging problems and to learning more about different research horizons.
Mobile antibiotic resistome in wastewater treatment plants revealed by nanopore metagenomic sequencing
In this study, we combined Oxford Nanopore and Illumina metagenomics sequencing to comprehensively uncover the resistome context of influent, activated sludge and effluent of three wastewater treatment plants (WWTPs) and simultaneously track the hosts of antibiotic resistance genes (ARG). The results showed that most of the ARGs detected in all compartments of the WWTPs were carried by plasmids. Transposons and integrons also showed higher prevalence on plasmids than on the ARG-carrying chromosome. Notably, integrative and conjugative elements (ICEs) carrying five types of ARGs were detected, and they may play an important role in facilitating the transfer of ARGs, particularly for tetracycline and macrolide-lincosamide-streptogramin (MLS). A broad spectrum of ARGs carried by plasmids (29 subtypes) and ICEs (4 subtypes) was persistent across the WWTPs. Host tracking showed a variety of antibiotic-resistant bacteria in the effluent, suggesting the high potential for their dissemination into receiving environments. Importantly, phenotype-genotype analysis confirmed the significant role of conjugative plasmids in facilitating the survival and persistence of multidrug-resistant bacteria in the WWTPs. At last, the consistency in the quantitative results for major ARGs types revealed by Oxford Nanopore and Illumina sequencing platforms demonstrated the feasibility of nanopore sequencing for resistome quantification. Overall, these findings substantially expand our current knowledge of the resistome in WWTPs, and help establish a baseline analysis framework to study ARGs in the environment.
Dr Tong Zhang is a Professor in the Environmental Biotechnology Laboratory in Department of Civil Engineering, and an Honorary Professor in the School of Public Health, at The University of Hong Kong. He received his Bachelor and Master’s degrees in Environmental Science and Engineering from Nanjing University, and his PhD from The University of Hong Kong. His research interests include environmental bioinformatics, omics technologies, anaerobic digestion and bioenergy from wastes/wastewater, biological wastewater treatment (N removal and P recovery), biodegradation of emerging pollutants (antibiotics, PPCP and EDCs) and antibiotic and antibiotic resistance genes. He has published over 200 peer-reviewed papers, and has more than 14, 000 citations and an H index of 68 on Google Scholar. He is an associate editor of Microbiome and Applied Microbiology and Biotechnology and had served as an advisor for Beijing Genomics Institute on Environmental Microbiology and Biotechnology from 2011 to 2014. He was Yi Xing Chair Professor of Nanjing University from 2013 to 2016, and currently is a distinguished visiting professor of Southern University of Science and Technology in China. He got First-Class Award in Natural Science of China Ministry of Education in 2015, Second-Class Award State Natural Science Award of China State Council in 2016, and Outstanding Research Student Supervisor Award of HKU in 2017. He is listed as one of the Highly Cited Researchers by Clarivate in 2018.
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.
Nanopore sequencing of the CYP2D6 pharmacogene
The accurate genotyping of CYP2D6 is hindered by the very polymorphic nature of the gene, high homology with its pseudogene CYP2D7, and the occurrence of structural variations. Using the GridION nanopore sequencer, we sequenced 32 samples covering various haplotypes of CYP2D6, including four samples with gene duplication, over two sequencing runs. The haplotypes of 26 samples could be matched accurately to known alleles or subvariants, while the remaining 6 samples had either novel variants or variant patterns not matched to the current PharmVar CYP2D6 haplotype database. Small insertions/deletions associated with several key haplotypes were detected accurately, and five novel variants not yet catalogues in PharmVar were reported. Allele duplication could be determined by analyzing the allelic balance between the sample haplotypes. Nanopore sequencing of CYP2D6 offers a high throughput method for genotyping, accurate haplotyping, and detection of new variants and duplicated alleles.
Yusmiati is currently a PhD Candidate at the Gene Structure and Function Lab in the Department of Pathology and Biomedical Science at the University of Otago, Christchurch. Her research focus includes application of nanopore sequencing and other sequencing methods in pharmacogenetics and adverse drug reaction. She holds a master’s degree in biomedical science from the University of Hasanuddin in Indonesia and has previously worked in a molecular diagnostic Lab in Jakarta, Indonesia.