Join us to hear the latest research from the Nanopore Community
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Sequencing and assembling the mega-genomes of mega-trees: the giant sequoia and coast redwood genomes
Johns Hopkins University
The giant sequoia tree (Sequoiadendron giganteum) and the coast redwood (Sequoia sempervirens) are two of the largest living organisms on the planet. A redwood is the world’s tallest tree at 115.7m (379.7 ft), and a sequoia is the world’s largest, at 1487 cubic meters. These trees also have giant genomes, at 8.2 Gb for sequoia and 26.5 Gb for redwood. Here I’ll describe our successful sequencing and assembly of a 1360-year-old sequoia tree (the tallest known sequoia tree on Earth, at 96.3m) which has produced the largest scaffolds of any genome project ever attempted, including telomere-to-telomere scaffolds for most of the 11 chromosomes. I’ll also describe our near-complete work on the genome of a 1390-year-old coast redwood tree. Both projects used a combination of short reads and long Oxford Nanopore reads for initial assembly, and Hi-C linked reads for scaffolding.
Steven Salzberg has been conducting research in genomics and computational biology for 25 years. In his early work at The Institute for Genomic Research, he developed novel computational methods for gene finding and contributed to many landmark genome projects, including the Human Genome Project and the genomes of dozens of bacteria, plants, and animals. He co-founded the Influenza Genome Sequencing Project in 2003, the first large-scale genomic survey of the influenza virus, and he led the team at TIGR that analysed the genome of the anthrax bacteria used in the 2001 anthrax attacks. Beginning in 2008, Salzberg and his students introduced several pioneering, highly efficient systems for analysis of next generation sequencing reads, including the Bowtie, Tophat, and Cufflinks systems, which were adopted by thousands of labs around the world. His lab members subsequently developed the HISAT and StringTie systems, even faster solutions to alignment and assembly for RNA sequencing experiments. Salzberg has authored or co-authored over 250 scientific publications that have garnered over 180,000 citations, and his h-index is 136. He is a member of the American Academy of Arts and Sciences, and a Fellow of AAAS and ISCB. He also writes a widely read column at Forbes that focuses on science and pseudoscience.
Oxford Nanopore Technologies
Sissel Juul joined Oxford Nanopore in the summer of 2014 to lead the company’s Genomic Applications group, setting up a lab in New York City. Recently, the Genomic Applications group has expanded, with the opening of a second lab, in the San Francisco Bay Area. These teams utilize the unique strengths of Oxford Nanopore technologies to showcase high-impact biological applications both independently, as well as with external collaborators and Oxford Nanopore customers. This leads to scientific papers, posters, and presentations at conferences. Prior to joining Oxford Nanopore, Sissel did her postdoctoral research at Duke University, NC, and has a PhD in molecular biology and nanotechnology from Aarhus University, Denmark.
Understanding genetic variation in cancer using targeted nanopore sequencing
Cold Spring Harbor Laboratory
Shruti received a B.Tech. in Genetic Engineering from SRM University, India in 2012 and went on to earn an M.Sc. in Molecular Biology and Human Genetics from Manipal University, India, in 2015. As part of her master's studies and following receipt of that degree, Shruti worked at Emory University on understanding the genetic basis of complex psychiatric disorders such as PTSD. She is currently a PhD candidate at Stony Brook University working on her dissertation research in Dr. W. Richard McCombie's lab at Cold Spring Harbor Laboratory. Her current focus is on developing targeted sequencing strategies using nanopore to resolve complex genomic regions associated with cancer.
Amplicons to whole genomes: clinical sequencing using nanopore technology
University of Birmingham
Andrew Beggs is a Reader in Cancer Genetics & Surgery at the University of Birmingham and a Consultant Colorectal Surgeon at the Queen Elizabeth Hospital Birmingham in the United Kingdom. He currently holds a Cancer Research UK Advanced Clinician Scientist fellowship and is a Fellow of the Alan Turing Institute. He runs a mixed wet/dry lab using genomics and patient derived disease models to answer translational research questions in cancer and immunology.
Measuring response to radiation exposure in human blood by long read nanopore sequencing
Public Health England
Christophe Badie leads the Cancer Mechanisms and Biomarkers Group of Public Health England’s Radiation Effects Department, which focuses on trying to better understand the mechanisms by which acute or protracted ionising radiation exposure, either of natural or medical origin, interact and affect cells and individuals in terms of inter-individual radiation sensitivity and susceptibility to long term effects such as radiation-induced cancer, particularly leukaemia. The group has developed new biomarkers of radiation exposure in human blood which are now validated in vivo in humans, carry out research in cancer genomics studying mutational signatures of ionizing radiation in second malignancies and study the effects of radiation exposure on the immune system, all also relevant to space exposure and astronauts.
Dr. Badie has an established reputation in the international radiation research field, committee member of the Association for Radiation Research (ARR), member of the European Radiation Research Society and the Research Orientation of the French Institute of Radioprotection and Nuclear Safety (IRSN) Committee, member of the NATO Research Task Group (RTG) Human Factors and Medicine Panel (HFM) HFM-291: Ionizing Radiation Bioeffects and Countermeasures and Member of the scientific council of The International Association of Biological and EPR Radiation Dosimetry (IABERD).
Nanopore RNA-Seq to HLA genotype and correlate donor HLA expression with flow cytometric crossmatch results
University of North Carolina
Transplant centers are increasingly using virtual crossmatching (VXM) to evaluate recipient and donor compatibility. However, the current state of VXM fails to incorporate donor HLA expression, despite numerous studies that demonstrated HLA expression impacts crossmatch outcomes. RNA-Seq for HLA enables simultaneous determination of HLA genotyping and relative HLA expression. Ultimately the RNA-Seq needs to be faster to incorporate into the VXM process. However, to demonstrate feasibility, nanopore-based RNA-Seq was evaluated for HLA genotyping and HLA class I expression. Nanopore RNA-Seq yielded 100% accuracy for HLA-A and HLA-B but 83.3% accurate for HLA-C. The decreased accuracy was attributed to low expression of HLA-C transcripts. The pattern of expression was relatively consistent, HLA-B>-A=-C. The impact of donor HLA expression on crossmatch outcomes was evaluated using serum containing a single donor-specific antibody (DSA). There was a correlation between the donor HLA expression to which the DSA was against and FCXM median channel shifts.
Dr. Weimer received his Ph.D. in Microbiology & Immunology from Wake Forest University 2009 with a focus on vaccine development. After completing two years of a basic immunology post-doc, Dr. Weimer transitioned to clinical immunology with a fellowship under Dr. John Schmitz at UNC Chapel Hill for two years. Dr. Weimer led the project which enabled the HLA laboratory at UNC to become one of the first hospitals to receive accreditation for HLA typing by NGS. In 2014, Dr. Weimer completed his training and stayed at UNC Chapel Hill as an Assistant Professor in the Department of Pathology & Laboratory Medicine, Director of Molecular Immunology, Associate Director of HLA, Immunology, and Flow Cytometry laboratories for UNC Hospitals. His research focuses on molecular diagnostic approaches for HLA genotyping and understanding solid organ transplant rejection.
The complete linear assembly and methylation map of human chromosome 8
University of Washington
Obtaining a complete high-quality, sequence-resolved human genome is essential to understand the genetic basis of human health and disease. Recent efforts to assemble human chromosomes from telomere to telomere have led to the successful assembly of the X chromosome and draft assemblies of many others (Miga, Koren et al., unpublished). However, the assembly of a human autosomal chromosome has not yet been completed. Here, we utilize a combination of long-read sequencing technologies, including Oxford Nanopore Technologies and Pacific Biosciences (PacBio), as well as a novel sequence assembly algorithm we developed, to complete the linear assembly of human chromosome 8. Specifically, we generate 20-fold coverage of ultra-long-read Oxford Nanopore data (16.6X > 100 kbp) and over 100-fold coverage of PacBio long-read data from the human hydatidiform mole, CHM13. We barcode each ultra-long Oxford Nanopore read with a set of singly unique nucleotide k-mers (SUNKs), which occur only once in the human genome and distinguish regions of the genome from one another. Then, we apply a novel assembly algorithm to link the barcoded Oxford Nanopore reads together, as well as a segmental duplication assembly algorithm to resolve sequence collapses, resulting in the linear assembly of the chromosome 8 centromere and two major segmental duplication blocks on the p- and q-arms. Using 24X PacBio HiFi (high-fidelity) reads, we polish each subassembly and incorporate these into the CHM13 assembly of chromosome 8. We are validating the assembly with several orthogonal technologies, including BioNano Genomics, Strand-seq, targeted BAC sequencing, and HiFi reads. Using the polished chromosome 8 assembly, we identify methylated DNA bases, creating a chromosome-wide methylation map. We find that methylated cytosines are specifically enriched at the pericentromeric transitions, consistent with the repressed gene expression reported in these regions. Together, our work reveals the complete linear sequence of chromosome 8 and provides a framework for understanding chromosome-level biology.
Glennis Logsdon is a postdoctoral research fellow in Evan Eichler’s laboratory at the University of Washington where she studies the sequence, variation, and function of human centromeres. She recently contributed to the first telomere-to-telomere assembly of a human chromosome, chromosome X, through the contribution of 14X ultra-long Oxford Nanopore reads with a read N50 near 150 kbp. Using these and other ultra-long read datasets, she is now working to complete the first telomere-to-telomere assembly of a human autosome, chromosome 8. The draft assembly of this chromosome reveals the linear sequence and organization of the 2 Mbp centromere as well as the 6 Mbp segmental duplication block, defensin, both of which have challenged previous efforts to elucidate their structure. Additionally, it uncovers the methylation status of these never-before-seen repeat regions. In the future, Glennis aims to lead an independent research program that builds upon her experience in genomics, synthetic biology, and genome engineering to understand the complex and repetitive regions of the human genome.
Genomes from metagenomes: assembling bacterial genomes with nanopore sequencing
The human gut microbiome is comprised of trillions of microbial cells that rapidly adapt to their environments, acquiring, losing, and transferring genetic material in response to selective pressures. However, the study of the microbiome has largely been limited to taxonomic classification and community composition, as high-quality genome assembly from complex communities is often untenable with short read sequencing. Advances in long read sequencing have allowed us to bypass many of these previous limitations and assemble more contiguous microbial genomes. We have demonstrated the application of nanopore sequencing for the generation of circularized, finished bacterial genomes for organisms that lack available short read reference genomes. This approach is able to place repeated elements in the proper genomic context and provide insight into structural variation in microbes, allowing us to move beyond taxonomic composition and towards functional characterization.
Dylan received a B.S. in Bioinformatics from Davidson College in 2018 and is currently a PhD student at Stanford University. She is working on her dissertation research in Dr. Ami Bhatt’s lab, which focuses on the application and development of new genomic and bioinformatic tools to understand the dynamics of the gut microbiome and how the microbiome can impact human health and disease. Dylan’s focus in on applying nanopore sequencing technology to improve microbial genome assembly and resolve circularized bacterial genomes, allowing for insight into genomic elements that often remain unassembled with short read metagenomic approaches.
Evaluation of the Oxford Nanopore MinION sequencing device for routine diagnostic testing and genotyping in a public health laboratory
New York State Department of Health
Next-generation sequencing technologies are being widely adopted as a tool of choice for diagnostic and outbreak investigation in larger public health laboratories. However, costs of operation remain major hurdles for laboratories with limited resources to adopt this technology. Using Mycobacterium tuberculosis as an example, we are evaluating the feasibility of using MinION whole-genome sequencing data for species identification, in silico spoligotyping, accuracy of detecting mutations associated with antimicrobial drug resistance, and phylogenetic analysis to discover transmission routes amongst patients. The results are then compared to those obtained with our current clinically validated Illumina MiSeq sequencing workflow. Here I present our results and findings of our ongoing side-by-side real-time comparison of MiSeq and MinION sequencing data for all patient samples received weekly since July 2019. Our preliminary assessment indicates that the MinION platform can achieve similar levels of genotyping and phenotypic resistance predictions than those of the Illumina MiSeq, at very competitive costs per sample.
Pascal Lapierre has been a research scientist in the bioinformatics core of the New York State Department of Health at the Wadsworth Center since 2013. Dr. Lapierre has more than 12 years experience in bioinformatics and genomic analyses of large-scale sequencing data from environmental and clinical sources. He is also a trained biologist with wet lab experience, and knowledge of infectious diseases and molecular evolution. He routinely analyzes next-generation sequence data from a variety of samples, such as bacterial pathogens, parasites, viruses and human subjects. These analyses provide valuable information for the accurate detection of outbreak sources, resolution of transmission pathways, improved resistance prediction, disease diagnostics and drug development. He has developed several bioinformatics pipelines currently used in NYSDOH clinical laboratories to direct or confirm ongoing epidemiological investigations, communicate resistance prediction profiles to clinicians, and perform species identification and genotyping. He also directs his own research on applying new technologies to clinical diagnostics.
Harnessing long-read sequencing for antibiotic discovery
Most of our currently available antibiotics were initially identified from soil bacteria. Whole-genome sequencing of soil isolates has revealed the potential of many of these organisms to produce over 20 secondary metabolites, many of which are unknown and could be developed into novel antibiotics. Due to their large size (>8 megabases), high GC content (~70%), and repetitive elements, these genomes are inherently challenging to resolve. Gene clusters involved in the production of antibiotics often span over 80 kilobases in length and can be split across contigs in assemblies produced through short-read sequencing, thus limiting our ability to predict the products of biosynthesis. We are harnessing long-read sequencing from Oxford Nanopore Technologies to generate complete genomes of soil bacteria and predict biosynthetic gene clusters. Uncharacterized clusters are chosen for genetic engineering and expression in production-optimized bacteria, followed by the purification of new compounds. Whole-genome sequencing with Nanopore will lead to the discovery of new antibiotics to replenish our arsenal of therapies for multi-drug resistant bacterial infections.
Allison received a B.Sc. in Biology at the University of Waterloo in 2016 before joining Dr. Gerry Wright’s research group at McMaster University in Hamilton, Ontario. As a Ph.D. student, she is working on developing a targeted capture approach to identify antibiotic resistance genes from both clinical and environmental samples. Outside of her main research interests, she is applying both short- and long-read sequencing to various research goals within the Wright Lab. This includes characterizing isolates from a collection of over 10,000 bacteria to predict and identify new antibiotic compounds.
Metagenomic nanopore sequencing for diagnosis of orthopaedic device-related infection
University of Oxford
Teresa is a senior laboratory scientist with the Modernising Medical Microbiology research group at the University of Oxford. The group aims to revolutionise diagnosis and management of infectious diseases in the National Health Service (NHS), in terms of personalised antibiotic treatment, continuous monitoring for outbreaks and discovery of new resistance mechanisms.
Teresa’s current research interests lie in the ability to utilise molecular techniques to improve the diagnosis of bacterial infections. She has used both short and long read whole genome sequencing technologies to identify pathogenic bacteria directly from clinical samples, without the need for an initial culture step. Her current research uses a metagenomic sequencing approach to identify and characterise pathogenic organisms causing orthopaedic device-related infections, and more recently to identify Neisseria gonorrhoeae directly from urine.
Teresa received her BSc and PhD from the University of Bath. She is also a lecturer in genetics at the Institute of Human Sciences and completed previous postdoctoral research at the Wellcome Trust Centre for Human Genetics, both at the University of Oxford.
Advances in Arachnid genomics using the Oxford Nanopore MinION
University of Maryland, Baltimore County
Sarah received her undergraduate degree in Biology in 2008, and a Masters in Entomology in 2011 from Clemson University, USA, before completing her Ph.D. in Biology from Virginia Tech, USA in 2015. During her postdoctoral fellowship at the US Army Research Laboratory, she studied the genetics of spider’s silks. She is currently an Assistant Research Scientist at the University of Maryland Baltimore County where she is investigating the genomics of parthenogenetic harvestmen.
Decoding the function(s) of m6A modifications on viral RNAs
New York University School of Medicine
Daniel P. Depledge
The RNA modification N6-methyladenosine (m6A) is a fundamental regulator of RNA biology that has been shown to play important proviral or antiviral roles in the life cycles of a growing number of viruses. Given the extremely compact nature of dsDNA viral genomes and the prevalence of many overlapping RNAs, identifying m6A modifications at the level of distinct RNA isoforms remains a significant challenge.
We have previously demonstrated how the application of nanopore direct RNA sequencing to primary cells infected with different dsDNA viruses (including herpesviruses and adenoviruses) enables us to examine splicing patterns, transcription initiation, and polyadenylation. To this we now add the ability to identify putative m6A modifications at nucleotide resolution while, crucially, distinguishing between overlapping RNA isoforms that are differentially m6A modified.
In addition to providing a technical overview of our approach and a critical discussion of sensitivity, specificity, and reproducibility, we will also share data on how manipulation of the methyltransferase complex can impair and enhance viral infections by influencing diverse aspects of RNA biology including splicing, read-through transcription, stability, localization, and the host interferon response.
Dan is an Assistant Professor at the New York University School of Medicine, currently setting up his first group. His research interests encompass transcriptional regulation, host-pathogen interactions, systems virology, and evolution. The primary focus of his research is on understanding the regulation of transcription in herpesviruses during lytic, latent, and reactivating infections – as viewed through both wet- and dry-lab approaches. He received a PhD in molecular parasitology from the University of York in 2009 and subsequently defected to virology, gaining postdoctoral experience at the Wellcome Trust Sanger Institute, University College London, and the NYU School of Medicine.
The Jackson Laboratory
Rachel Goldfeder is a Computational Scientist on the Genome Technologies team at The Jackson Laboratory for Genomic Medicine. Her research interest is in using novel sequencing approaches to aid disease understanding, diagnosis, prognosis, and treatment. Rachel holds a BS in Biomedical Engineering from Washington University in St. Louis and a PhD in Biomedical Informatics from Stanford University.
Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia
University of California, Santa Cruz
SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by SF3B1 mutations using short-read sequencing are known, identifying the full-length isoform changes with nanopore sequencing may better elucidate the functional consequences of these mutations. We have sequenced cDNA from CLL samples with and without SF3B1 mutation with the PromethION giving a total of 149 million pass reads. We then developed FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify expressed transcripts. FLAIR differential splicing analysis of the data recapitulated known effects of 3’ splice site changes caused by SF3B1 mutations. Notably, we observed a strong downregulation of a set of IR events associated with SF3B1 mutation more confidently observed with nanopore reads, a finding which was further supported with the reanalysis of short reads. Our work demonstrates the utility of nanopore sequencing for cancer and splicing research.
Alison Tang is a graduate student in the bioinformatics program at the University of California, Santa Cruz, advised by Dr. Angela Brooks. Alison's work is focused on improving the resolution of the transcriptome, particularly that of human disease, using nanopore sequencing technology. The length of nanopore native RNA and cDNA reads allows for a more accurate look at exon connectivity; Alison has leveraged this to develop computational methods for building high-confidence isoform references and analyzing alternative isoform usage. Alison has used nanopore sequencing to investigate the impact of cancer-associated mutation in splicing factor SF3B1 on RNA splicing at an isoform level. Prior to her graduate studies, Alison attended UC Berkeley where she studied molecular and cell biology and learned how to code.
PCR-free transposon sequencing (TnSeq): dCas9/Cas9-mediated transposon enrichment
University of Arkansas for Medical Sciences
Transposon sequencing (TnSeq) is a genome-wide screen of microbial libraries with transposon insertions. The current TnSeq protocol relies on polymerase chain reaction (PCR) amplification of the transposon junctions to identify and enumerate insertions. PCR introduces bias that may distort our interpretation of the results. This necessitates the development of alternative PCR-free sequencing approaches that quantitatively measure insertion frequencies. We conducted a TnSeq analysis using a highly saturated library in the bacterial pathogen, Staphylococcus aureus. To eliminate the PCR step, we adapted the Cas9-Nanopore target enrichment protocol to sequence the TnSeq library by pulling down the transposon-genome junctions using a biotinylated deactivated Cas9 (dCas9) and later cleaving the transposon with an active Cas9. We achieved up to 48% enrichment of the transposon when applying this method. Our findings may further strengthen the robust TnSeq screen as the elimination of PCR bias may yield more specific hits with minimal false positives.
Duah Alkam received her B.S. and M.S. from The Hebrew University of Jerusalem before joining Dr. David Ussery and Dr. Mark Smeltzer’s groups as a PhD student at The University of Arkansas for Medical Sciences. Her research focuses on using transposon sequencing to identify Staphylococcus aureus genes that contribute to the pathogenesis of osteomyelitis (bone infection). Her project merges a robust in vivo model of Staphylococcus aureus osteomyelitis with genomic analyses which she implements under the direction of Dr. Piroon Jenjaroenpun. During her tenure as a graduate student, she helped develop a pipeline to rapidly sequence and characterize mumps virus genomes using Oxford Nanopore Technologies. She, along with a team led by Dr. Se-Ran Jun, implemented this pipeline on a mumps virus strain that caused an outbreak in the state of Arkansas in 2016.
Nanopore PromethION sequencing of the Genome in a Bottle (GIAB) HG002 sample
Long read sequencing technologies have the potential of resolving complex genomic features, including repetitive regions and structural variants. Oxford Nanopore Technologies provides competitive long-read sequencing platforms that have a relatively low cost. Furthermore, nanopore sequencing of human genomes has become easier with the launch of the Oxford Nanopore PromethION platform. However, some of the challenges of this third-generation sequencing technology are i) the relatively low raw read accuracy, and ii) the lack of a well characterized bioinformatic pipeline for data processing. Here we show that a single PromethION flow cell can generate enough data to resolve the genomic features as described above. We sequenced the Genome in a Bottle (GIAB) sample HG002/GM24385 using the LSK-SQK109 ligation kit and a single PromethION flow cell with R9.4 pores. The run generated 95.47 Gb of high accuracy basecalled sequencing data. This is equivalent to a 23-fold mean genome coverage with over 96% of bases at a read depth of 10 or higher. Benchmarking structural variants against the GIAB curated truth set v0.6 of sequence-resolved tier 1 and complex tier 2 variants of GM24385/HG002 revealed that our in-house preparation is comparable to the GIAB 52x state-of-the-art genome. A precision of 0.9421 and recall rate of 0.9571 was achieved (F1 of 0.9469). Moreover, despite ranking second only to the recent PacBio pbsv 2.2.1 - Sequel2 GIAB dataset in tier 1 variant calling in cross-technology comparisons, our in-house sample ranked first in identifying tier 2 variants, suggesting Oxford Nanopore long-read technology may help resolve more complex structural variants than other methods. Thus, with sufficient coverage, a single PromethION flow cell may perform equal or better for analysis of a human genome than other commonly used next generation or third generation sequencing platforms. Furthermore, PromethION sequencing brings the total cost of consumables down to $2,175 per human genome.
Dr. Sarah Reiling joined the McGill University Genome Sciences Centre in 2019 and oversees the Centre’s nanopore sequencing platforms, working on various international projects. Prior to working at McGill, Dr. Reiling set up and performed the first nanopore sequencing runs at the Health Canada Food Directorate, Bureau of Microbial Hazards, where she worked as a postdoctoral fellow. She has given nanopore workshops at Health Canada and McGill University, and presented her work at international conferences.
Dr. Reiling’s main focus is on human genetics and human pathogenic parasites. During her career, she worked on multiple international projects in Asia, Africa, Europe, and North America. To date, her primary focus is on benchmarking structural variants in human genomes using nanopore technology.
Full length cDNA sequencing coupled to time-of-day sampling enables enhanced gene prediction in the fastest growing plant on Earth
J. Craig Venter Institute
While long read nanopore sequencing has ushered in a new era of contiguous genome assemblies, accurate and complete gene predictions remain a challenge especially for unique species in underrepresented regions of the tree of life. Nanopore sequencing enables full length cDNA (FL-cDNA) sequencing that provides essential empirical evidence of intron-exon structures and alternative splicing, which compliments ab initio efforts to define gene models. Here we coupled time-of-day (TOD) sampling with FL-cDNA sequencing to improve the sensitivity and precision of gene calls in Duckweed. The Greater Duckweed, Spirodela polyrhiza is re-emerging as a model plant due to its extremely fast growth rate, aquatic lifestyle, small genome, minimal set of genes, transformation system, and reduced body plan. These results highlight the utility of using FL-cDNA to accurately annotate genomes and determine gene expression patterns.
Dr. Todd Michael is Professor and Director of Informatics at the J. Craig Venter Institute (JCVI) in San Diego, CA USA. Dr. Michael has led academic and commercial genome centers, and currently directs the JCVI Sequencing Core. In addition, his research group is interested in leveraging sequencing technologies and informatics to understand how information is stored in genomes, such as genome architecture, gene and repeat content, and epigenomic state.
Johns Hopkins University
Mihaela Pertea is an Associate Professor in the Department of Biomedical Engineering at Johns Hopkins University. She received her B.S. and M.S. degrees in Computer Science from University of Bucharest in Romania, and her Ph.D in Computer Science from the Johns Hopkins University School of Engineering. Dr. Pertea’s work in computational biology draws upon techniques and data from multiple disciplines, including computer science and molecular biology, genetics, biotechnology, and statistics. Her work has focused on computational gene finding and sequence pattern recognition and she has developed several open-source gene finders that were used for the annotation of the genomes of Plasmodium falciparum (malaria parasite), Arabidopsis thaliana, rice, Aspergillus fumigatus, Cryptococcus neoformans, and others. A major focus of her current research is on developing innovative and efficient methods to analyze large DNA and RNA sequence data in order to provide a genome-scale understanding of cellular function. Dr. Pertea believes that the principled use of algorithms from other fields, adapted to the problems of computational biology and coupled with careful software engineering and high-performance computing, has the potential to make a significant impact in the life sciences. She has published over 50 scientific papers that have received more than 27,000 citations to date.
Long reads reveal small scale genome structural variations in Allo-tetraploid Canola
Justus Liebig University
Harmeet Singh Chawla
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.
Detection of RNA modifications and structure using nanopore sequencing
New York Genome Center
Chemical modifications decorate several major classes of RNA including tRNA, rRNA and mRNA, providing structure and functionality beyond that found in the four canonical ribonucleosides. Ribosomal RNA in particular is heavily modified, many positions at near stoichiometric levels. Here we utilize direct RNA nanopore sequencing to qualitatively and quantitatively characterize modifications in rRNA from E. coli and S. cerevisiae. We find that modified nucleosides cause perturbation of ionic flow through the protein nanopore that is detectable in the raw current measurements. In addition, we describe and characterize a novel modality of measurement using protein nanopores characterized by alteration in the time domain of nucleic acid translocation through the pore in a modification-dependent manner. Furthermore, we exploit this effect to profile RNA structure by exogenously labelling flexible nucleotides in a folded RNA using a novel chemical probe. Long read information obtained by sequencing RNA directly enables the detection of multiple modifications at the single molecule level, providing information on phasing of modifications and expanding the ability to decipher full-length RNA structure.
William Stephenson is a Senior Research Engineer in the Technology Innovation Lab at the New York Genome Center. His research interests include single molecule biophysics, microfluidics and single cell genomics. William holds a BS in Physics from Drexel University and a PhD in Nanoscale Engineering from the University at Albany.
A targeted nanopore sequencing based test for the rapid diagnosis of drug-resistant TB
Quadram Institute Bioscience
Tuberculosis (TB) is one of the top 10 causes of mortality globally and the leading cause from a single infectious agent, causing approx. 1.6 million deaths in 2017. Drug-resistant TB (DR-TB) threatens global TB care and prevention and is a major public health concern in many countries. Worldwide in 2017, over half a million people developed DR-TB, of which 8.5% were estimated to have extensively drug-resistant TB (XDR-TB). Rapid and accurate diagnosis of TB and DR-TB, followed by provision of appropriate treatment, prevents deaths, limits ill-health and stops further transmission of infection to others.
We have developed a targeted nanopore sequencing test for the rapid diagnosis of DR-TB directly from sputum samples. The test targets 16 genes, which cover all the high/moderate confidence single nucleotide polymorphisms (SNPs) associated with drug resistance and can identify MDR- and XDR-TB directly from sputum within 6 hours. The nanopore TB test is currently being assessed in a blinded analytical testing study with FIND. In this talk I will describe the development and optimisation of the method.
Dr. O'Grady gained his B.Sc. in Microbiology, his M.Sc. (Res) in infectious diseases diagnostics and his Ph.D. in the molecular diagnosis of pathogens in food all at the National University of Ireland Galway (NUIG). He remained at NUIG for his first post-doc, continuing his research in food microbiology. This was followed by a two-year stint in industry (Beckman Coulter) developing real-time PCR based tests for infectious diseases including tuberculosis (TB). Dr O’Grady then returned to academia, taking up a post-doc position at University College London on TB diagnostics. In January 2013 he was appointed Assistant Professor in Medical Microbiology at UEA, was promoted to Associate Professor in August 2016, and in April 2018 also took the role of Research Leader at Quadram Institute Bioscience. His research continues to focus on the rapid molecular diagnosis of pathogens with the aim of translating this research broadly, in different sectors and diseases, to maximise community/patient benefit.
Real-time assembly using nanopore sequencing data for microbial communities
The University of Queensland
Real-time sequencing output is an interesting property that makes Oxford Nanopore platforms stand out from others. In this presentation, I will describe a set of our bioinformatics tools for the task of completing microbial genome assembly in real-time. Basically, the input stream of long-read data is taken as input to connect and finish a pre-assembly structure, such as Illumina assembly contigs or assembly graph, by a streaming algorithm that allows the process to happen abreast with the sequencing operation. By applying such quick-response pipeline, the assembly results can be instantly reported and visualized without the need to wait until the nanopore run is finished.
Son Nguyen graduated with Honors at the University of Engineering and Technology, Vietnam National University Hanoi in Computer Science. Being oriented towards Life Science, Son completed a joint Masters degree of System Biology in Europe before heading to the University of Queensland, Australia for his Ph.D in July 2015. He has been working as a Postdoctoral researcher at the Institute for Molecular Bioscience, UQ since 2019.
Son’s research interests include computational biology and bioinformatics. Recently, his works involve the applications of Oxford Nanopore Technology sequencing platforms to assembly and analyze microbial genomes, focusing on the real-time function of the devices. He has developed and still working on the development of several streaming pipelines that could help reduce the turn-around time and resources compared to traditional approaches.
Selective single molecule sequencing and the assembly of human Y chromosomes
Universitat Pompeu Fabra
Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions, only a handful of species have their Y chromosome properly characterized. To date, only a single human reference quality Y chromosome, of European ancestry, is available because of a lack of accessible methodology. To facilitate the assembly of such complicated genomic territory, we developed a strategy to sequence native, unamplified flow sorted DNA on a MinION sequencing device. Our approach yields a highly continuous and complete assembly of the human Y chromosomes of diverse origins highlighting the complex inner structure of this chromosome. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800% thus allowing the chromosome scale analysis of human Y chromosomes. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes.
Dr. Marques-Bonet is the Principal Investigator of the group "Comparative Genomics" at the University Pompeu Fabra and Institute of Evolutionary Biology (UPF and CSIC) with a dual appointment at CRG/CNAG. He was awarded with a Howard Hugues Medical Institution (HHMI) Early International Career Award in 2017. His group is interested on characterizing in population genomics of non-human primates in detecting human specific genomics features and the evolution of epigenetics in humans and non-human primates.
The Jackson Laboratory
Partner-independent fusion gene detection by multiplexed CRISPR/Cas9 enrichment and long-read nanopore sequencing
Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment possibilities. The promiscuity of fusion genes with respect to partner choice and exact breakpoint-positions restricts their detection in the diagnostic setting. To accurately identify these gene fusions in an unbiased manner, we developed FUDGE: a FUsion gene Detection assay from Gene Enrichment. FUDGE couples target-selected and strand-specific CRISPR/Cas9 activity for enrichment and detection of fusion gene drivers - without prior knowledge of fusion partner or breakpoint-location - to long-read nanopore sequencing. FUDGE encompasses a dedicated bioinformatics approach (NanoFG) to detect fusion genes from nanopore sequencing data. Our strategy is flexible with respect to target choice and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in a single sequencing run. We demonstrate that FUDGE effectively identifies fusion genes in cancer cell lines, tumor samples and on whole genome amplified DNA irrespective of partner gene or breakpoint-position in 100% of cases. In summary, we have developed a rapid and versatile fusion gene detection assay, providing an unparalleled opportunity for pan-cancer detection of fusion genes in routine diagnostics.
Christina obtained her B.Sc. and M.Sc. in Biology at the University of Regensburg. She is currently completing her PhD at the UMC Utrecht and the NKI Amsterdam. She focuses on cancer genomics with the main goal to utilize structural variations present in cancer genomes for personalized cancer care. Together with her colleagues she uses the real-time and long-read capabilities of nanopore sequencing to develop assays aimed for clinical applications. So far, she has developed a targeted nanopore sequencing assay to rapidly detect fusion genes irrespective of fusion-partner and/or breakpoint-location aimed for diagnostic purposes. Furthermore, she is involved in the development of an assay to rapidly identify tumor-specific structural variation biomarkers with nanopore sequencing to facilitate immediate disease monitoring and minimal residual disease testing of cancer patients from liquid biopsies.
University of Liverpool
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
Nanopore Community Meeting in San Francisco 2018
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