Join us in New York this December to hear about the latest research from the Nanopore Community!
As well as plenary, breakout and lightning talks, you'll have the chance to get hands-on with nanopore technology in the live lounge and see it in action during the demos.
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.
The long and short of it
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.
The long and short of it
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.
Understanding genetic variation in cancer using targeted nanopore sequencing
Cold Spring Harbor Laboratory & Stony Brook University
Cancer cells acquire varied genetic aberrations many times through different degrees of genomic instability during tumorigenesis, evolution, and disease progression. Structural variations (SV), a hallmark of genomic instability in cancer, include insertions, deletions, duplications, inversions, or translocations, typically larger than 50bp, that can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types, including but not limited to, high-grade serous ovarian, esophageal, neuroblastoma, small-cell lung cancer, pancreatic cancer, and triple-negative breast cancers. These studies highlight the pervasive nature of SVs and the importance of studying them in the cancer genome. The discovery of genetic variants is highly dependent on the methods employed to detect and measure them. Standard tools for SV detection such as microarrays and cytogenetic methods are low resolution and do not offer nucleotide-level information. High resolution next-generation sequencing (NGS) platforms have revolutionized cancer genomics and have been instrumental in characterizing single-nucleotide variants (SNVs) and small variants. However, NGS is partially blind to SVs as it relies on the alignment of millions of short reads to produce a final genome sequence, effectively losing long-range information, lacking sensitivity, and exhibiting very high false positive rates (up to 89%) depending on the type of SV. Long read, single-molecule sequencing methods, such as Oxford Nanopore Technologies, can address larger variations as they typically generate read lengths of tens of thousands of bases. In recent years, long-read sequencing strategies have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage, coupled with the high cost of whole genome long-read sequencing makes it infeasible to conduct large-scale genomic studies, limiting our understanding of the distribution and frequencies of SVs in the population. Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population. However, a lack of efficient long-read compatible targeting techniques makes it difficult to study specific regions of interest on existing long-read platforms. To address this, we are evaluating amplicon-based and CRISPR/Cas-based systems of targeted long-read sequencing to enrich for specific regions of the cancer genome (panel of >10 genes, including BRCA1 and BRCA2) in two breast cell lines – MCF 10A and SK-BR-3. Our goal is to develop a long-read breast cancer panel to facilitate large population SV analysis, which will help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest.
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
Nanopore sequencing has a number of advantages over short read sequencing, including enhanced structural variant detection and native methylation detection, in addition to the advantages of short reads in resolving typically difficult regions in the genome. In this talk, I will present three pieces of work on behalf of our laboratory. Firstly, I will describe the work we have been carrying out using nanopore sequencing to highly accurately type the Human Leucocyte Antigen (HLA) region to 4 field accuracy, as well as phase HLA and detect runs of homozygosity which have previously been extremely challenging using short read technologies. Secondly, I will describe the advances we have made in low pass whole genome copy number & detection of methylation using the Flongle on human cancer samples. Finally, I will present our experiences with long read sequencing of samples derived from the 100,000 Genomes project using the PromethION system, including a pipeline to call variants, SVs, CNAs and methylation in 24 hours. I hope to demonstrate that nanopore sequencing is an emerging technology with multiple applications in the clinical sphere.
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.
Generation of a new transcriptional radiation exposure signature in human blood using long-read nanopore sequencing
Public Health England
In the event of a large-scale event leading to acute ionising radiation exposure, high-throughput methods would be required to assess individual dose estimates for triage purposes. Blood-based gene expression is a broad source of biomarkers of radiation exposure which are of great potential to provide rapid dose estimates for a large number of individuals. Time is a crucial component in radiological emergencies and the shipment of blood samples to relevant laboratories is an issue. In this study, we have performed nanopore sequencing analysis to determine if the technology can be used to detect radiation-inducible genes in human peripheral blood mononuclear cells (PBMCs). The technology offers not only long-read sequencing but also a portable device which can help solve the sample shipment issues, providing faster results. For this aim, blood from nine healthy volunteers was ex vivo irradiated with a 2 Gy X-rays dose. Following PBMCs isolation, irradiated samples were incubated along with the controls for 24 h at 37°C. RNA was extracted, poly(A)+ enriched and reversed transcribed before sequencing. The data generated was analysed using a snakemake pipeline modified to handle paired samples. The sequencing analysis identified 46 differentially expressed genes (DEGs) which included 41 protein-coding genes, a long non-coding RNA and 4 pseudogenes, amongst which five of them have been identified as radiation responsive transcripts for the first time. The most significantly regulated genes after radiation exposure were APOBEC3H and FDXR, presenting a 25 and 28-fold change on average respectively, levels of transcriptional response comparable to results we obtained by quantitative polymerase chain reaction (qPCR) analysis. In vivo exposure analyses showed a transcriptional response to IR after 24 h post-exposure for both genes together with a strong dose-dependent response in blood irradiated ex vivo. Finally, extrapolating from the data we obtained, the minimum sequencing time required to detect an irradiated sample using APOBEC3H transcripts would be less than 3 minutes for a total of 50000 reads. Therefore, by future improving of sample processing and developing bioinformatic tools for specific transcript identification, nanopore sequencing technology could potentially help to develop a fast, in the field and real-time biodosimetry platform. In summary, our data show that nanopore sequencing can identify radiation-responsive genes and can also be used for identification of new transcripts.
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 analysis of orthopaedic device-related infection
University of Oxford
Prosthetic joint infections (PJI) are a potentially devastating and difficult to treat complication of joint replacement surgery. The current gold standard for microbiological diagnosis - culture of multiple periprosthetic tissue samples - is imperfectly sensitive, particularly in infection with fastidious organisms or following antimicrobial therapy prior to surgery. Additional diagnostic information can be obtained through sonication fluid culture of explants. We assessed whether nanopore metagenomic sequencing of DNA extracted from sonication fluid can provide a sensitive tool for diagnosis of PJI and provide sufficient data to make predictions regarding antibiotic susceptibilities. We demonstrate that nanopore metagenomic sequencing can provide informative diagnostic information in the case of PJI. Successful depletion of human DNA prior to sequencing improves the breadth and depth of coverage of the reference genome in the majority of cases, allowing detection of antimicrobial resistance determinants.
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
Oxford Nanopore technology is allowing for advances in arachnid genomic biology by providing both scaffolds for repetitious spider silk genes and long reads for the complex genomes of polyploid, parthenogenetic harvestmen. The aggregate gland protein glue which coatsnthe prey-capture threads of orb web and cobweb weaving spiders is encoded by members of the silk gene spidroin (spider fibroin) family. Spidroins are extremely large, highly repetitive genes that cannot be sequenced using short-read technology alone. We used the Oxford Nanopore MinION to discover for the first time, the complete sequences of two glue genes from the orb weaving spider Argiope trifasciata. These genes, one of which has a coding domain region of greater than 40kb, are the largest spidroins currently described. Our latest project uses nanopore technology to sequence the genome of the facultatively parthenogenetic Japanese harvestman Leiobunum manubriatum, which exhibits populations with diploid and tetraploid individuals. Whole genome sequencing on the MinION has yielded the first mitochondrial genome for the L. manubriatum family Sclerosomatidae and will be used to investigate genome evolution between cytotypes. Recent research suggests mitonuclear interactions reinforce reproductive mode, such that sequencing the challenging genomes of parthenogenetic taxa will facilitate further research on the evolution of sex.
Sarah received her undergraduate degree in Biology in 2008, and a Masters in Entomology in 2011 from Clemson University, USA, before 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.
Evaluating and comparing transcriptome sequencing approaches
The Jackson Laboratory
RNA sequencing provides insight into the molecules that are actively expressed in a given tissue type at a certain point in time. Traditional RNA sequencing approaches generate cDNA from RNA templates using reverse transcriptase, usually with PCR amplification. Therefore, traditional approaches are subject to biases that are associated with the reverse transcriptase enzyme and PCR and are not able to assess RNA modifications. Oxford Nanopore’s new direct RNA sequencing approach does not depend on reverse transcriptase or require PCR, allowing users to overcome biases typically associated with their use. Here, we evaluate direct RNA sequencing, PCR-free cDNA sequencing, and cDNA sequencing with PCR in terms of their yield, read lengths, read mappability, transcripts covered, and isoform definitions. We also compare each approach’s consistency across replicates. Finally, we show that RNA modifications can be readily detected from direct RNA sequencing datasets.
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.
Efficient and robust transcriptome reconstruction from long-read RNA-Seq alignments
Johns Hopkins University
RNA-sequencing technologies are a key component in characterizing the structural complexity and expression profiles of the set of RNA transcripts produced in a cell. While second-generation sequencers produce very large numbers of reads, their read lengths are typically quite short, in the range of 75-125 bp for most experiments. These short reads often align to more than one location, and also suffer the limitation that they rarely span more than two exons, sometimes making impossible to identify correctly all the isoforms of a gene. These issues can be alleviated by third-generation sequencing technologies such as those from Oxford Nanopore Technologies. These long-read technologies, which can produce read lengths in excess of 10,000 bp, offer the potential for large gains in the accuracy of isoform identification and discovery. To date, though, long reads have been challenging to be adopted for transcriptome assembly, in part because of their higher error rate which makes their alignment to a known reference genome difficult.
Recently we have developed StringTie, a genome-guided transcriptome assembler that runs much faster and provides more accurate overall results than similar competing methods. Here we present StringTie2, a major new release of the StringTie transcript assembler, which is capable of assembling both short and long reads. It also offers the ability to work with full-length super-reads assembled from short reads, which further improves the quality of assemblies. Our results on 33 Illumina RNA-seq datasets demonstrate that StringTie2 is more accurate than Scallop, the next-best performing transcriptome assembler of those currently available. The use of super-reads also consistently improves both the sensitivity and precision of StringTie2 assemblies. On multiple Oxford Nanopore long read datasets, StringTie2 on average correctly assembles many more transcripts and with substantially higher precision than FLAIR, a known pipeline to identify transcripts from long reads. StringTie2 is also faster and has a smaller memory footprint than all comparable tools.
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 allotetraploid canola
Justus Liebig University
Harmeet Singh Chawla
There is increasing evidence that genome structural variation (SV) contributes significantly to phenotypic variation for many important agronomic traits. Most crop plants derive from ancient or recent polyploidisation events and carry extensive SV, however difficulties in describing this kind of variation with common genetic marker systems or short-read DNA sequencing mean that SV has been largely neglected when it comes to explaining observable phenotypes. Accurate long-read sequencing provides new opportunities to detect SV at the scale of single-genes and associate such variants to traits. Here we describe the use of Oxford Nanopore sequencing for precise detection of small-scale SV and association with quantitative disease resistance and various other agronomically interesting traits in Brassica napus (canola, rapeseed), a recent allopolyploid species with a complex, rearranged genome which is today the world’s second most important oilseed crop. Whole genome sequencing was performed for a commercial cultivar along with two synthetic B. napus lines carrying interesting trait variation for breeding, in order to identify the distribution and frequency of large-scale and small-scale SVs and investigate associations with known QTL in crosses between these lines. We established efficient and fast protocols for high molecular weight DNA isolation and library construction that enabled high data yields with optimal sequence quality on the Oxford Nanopore MinION system. Although DNA quality in Brassica species is often poor due to high levels of secondary compounds in the leaves, our protocols enabled consistent yields of 10-12 GBp per nanopore flowcell, corresponding to around 8x – 10x coverage of the B. napus genome (1.3 Gb). Using this data, we were able to improve assembly accuracy of problematic, strongly homologous chromosome regions and to identify large-scale and small-scale SVs (down to single-gene level) associated with quantitative disease resistance and other traits. Reduced costs and improved data quality from long-read sequencing technologies provide new opportunities for analysis and gene discovery in polyploid crops where reference-based sequence mapping approaches fail to capture important SV.
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 microbial assembly using nanopore sequencing
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.
Nanopore ultra-long read sequencing provides insights into structural variation in cancer genome
The Jackson Laboratory
Genomic structural variants (SVs), the major hallmarks of cancer genomes, can promote tumor progression by perturbing gene structures and expression control, which contribute to tumor heterogeneity and evolution. Single-molecule DNA sequencing technologies offer significantly longer read lengths which can facilitate the detection and analysis of highly rearranged cancer genomes. In this study, we generated deep coverage of ultra-long reads from three glioblastoma (GBM) patient-derived neurospheres and presented the unbiased survey of full-scale SV landscapes with our customized SV caller Picky. We detected complex SVs with low allele frequency and provided SV with phasing information. Comparing the SVs in the primary and recurrent tumors from the same patients, we revealed rearrangement events associated with tumor relapse and characterized their genomic features associated with chromatin organization and transcriptional regulation. A complete understanding of the structure and distribution of SVs in cancer genome will empower the cancer research community to reveal mechanisms that induce genome instability, identify prognostic signatures of tumor progression and suggest targets for novel treatment strategies.
Liang Gong is a postdoctoral associate in the Genome Technologies group at The Jackson Laboratory for Genomic Medicine. His research interests the single molecule long-read sequencing technologies, genomic structural variations and the transcriptional regulation in cancer genome. Liang holds a BS in Biological Science and a PhD in Biochemistry and Molecular Biology from Huazhong Agricultural University.
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.
CRISPR/Cas9-mediated PCR-free targeting in nanopore sequencing: understanding RAS isoform mutations
University of Liverpool
The RAS proto-oncogenes are amongst the most frequently mutated in cancer, with specific activating mutations influencing clinical outcomes. HRAS, KRAS and NRAS have close sequence similarity, but their mutational profiles differ significantly, leading us to hypothesise that their susceptibility to mutagen targeting and repair is sequence context dependent. Mutations in these isoforms occur due the interaction of mutagen targeting and access to cellular repair mechanisms. We hypothesise that subtle nucleotide differences could influence mutation rates in the isoforms. To determine the role of sequence context in mutational spectra, we are developing a method for targeted sequencing of RAS genes using a combination of cas9 cleavage, PCR-free amplification and long read sequencing. PCR amplification methods for enrichment are length limited, modifications are lost, and amplicon biases can be introduced. Therefore, we are developing a method of PCR-free amplification and targeted sequencing of RAS genes in different mutagen contexts using the Oxford Nanopore Technologies MinION platform.
Jennifer Mitchell obtained her MBiolSci Integrated Master’s degree in Biological Sciences from the University of Liverpool. As a graduate she worked as an Associate Scientist at RedX Pharma working within the Biology Oncology group. She is currently completing her PhD at the University of Liverpool Institute of Translational Medicine under the supervision of Professor Ross Sibson and Professor Ian Prior. Her work involves using sequencing technologies to understand mutagenesis mechanisms in cancer. Her research utilises the CRISPR/Cas9 system for targeted nanopore sequencing of the RAS genes.
Targeted, high-resolution RNA sequencing of non-coding genomic regions associated with neuropsychiatric functions
Weill Cornell Medicine
The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimer's and Parkinson's disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ('RNA CaptureSeq') in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNA-seq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of non-coding transcription in human brain that can be used to connect neurological phenotypes with gene expression.
Dr Simon Hardwick is a postdoctoral researcher at Weill Cornell Medicine in New York City. The primary focus of his research is studying RNA isoform usage and splicing in the brain, with a view to better understanding neurodevelopment and neurodegeneration. His research involves a combination of innovative genomic technologies including single-cell RNA-seq, long-read sequencing and targeted RNA capture, as well as the development of accompanying bioinformatic tools. He is the recipient of an Australian NHMRC Early Career Fellowship. He received his PhD in genomics from UNSW Sydney in 2018, where he successfully led a project to develop an innovative framework for designing synthetic spike-in controls (‘sequins’) for next-generation sequencing assays.
Long-read genomes and transcriptomes on the cloud
Kiran V Garimella
Effective processing of long-read genomic and transcriptomic datasets involves many computationally expensive steps, spanning GPU-enabled base/modification calling, haplotype-resolved de novo assembly, assembly error correction, structural variant detection, novel transcript isoform identification, etc. The data storage and processing needs may be difficult to satisfy at scale using local high-performance compute facilities. Migration to the cloud poses significant implementation challenges, but if used effectively, can enable cost-effective access to a far greater set of computational resources than on-premises resources can supply. Here, we report on our ongoing efforts to develop effective, efficient, reproducible, and cloud-friendly pipelines for long-read genomic and transcriptomic analysis. We demonstrate this work on a variety of applications, including GridION-based cDNA sequencing of ~80 GTEx samples, haplotype-resolved assembly of mother/father/child trios, and identification of simple and complex SVs.
Kiran V Garimella is a senior computational scientist in the Broad Institute’s Data Sciences Platform (DSP) where he runs a new group dedicated to methods development and applications of long reads. Prior to this, Kiran attended the University of Oxford on a Wellcome Trust scholarship, completing a DPhil and post-doc as a member of the McVean lab. There he worked on integrating long-read data with short reads for improved genome assembly and complex structural variant discovery in hypervariable and repetitive regions of malaria genomes. Kiran has also spent the summer of 2018 as a visitor to the Simpson lab at University of Toronto getting hands-on experience with the Oxford Nanopore PromethION processing and examining long-read data on leukaemia genomes.
Detection and study of DNA damage-induced non-coding RNAs
IFOM - FIRC Institute of Molecular Oncology
Alessia di Lillo
We reported that RNA polymerase II is recruited to the exposed DNA ends of DNA double-strand breaks (DSBs) and synthesizes damage-induced long non-coding RNAs (dilncRNAs) (Francia et al, Nature, 2012; Michelini et al, NCB, 2017; Pessina et al, NCB, 2019). Others have reported that DNA damage can positively regulate transcription initiation and that neuronal activity induces DSBs within promoters of early-response genes required for their transcription. DSB-induced transcripts are very low abundant and difficult to detect by Illumina sequencing for reasons that remain unclear, while they are robustly detectable by single-molecule FISH and RT-qPCR. Using Oxford Nanopore technology, we performed direct cDNA sequencing which allowed the detection of transcripts emerging in proximity of the DSB site, compared with the control sample. This therefore seems to suggest that Oxford Nanopore technology is a promising approach that can help us to characterize better this new class of ncRNAs generated upon DNA damage.
Alessia di Lillo obtained her master’s degree cum laude in Biology from the University of Milano Bicocca in Italy. In 2016, she passed the entry exam at the European School of Molecular Medicine (SEMM) and she started her Ph.D. in Fabrizio d’Adda di Fagagna’s group at IFOM – FIRC Institute of Molecular Oncology – in Milan and is currently working on the link between DNA damage and transcription. In particular, she focusses on the characterization of damage-induced long non-coding RNAs generated at the site of damage, exploiting Oxford Nanopore technology, as it allows a single-molecule direct cDNA and RNA-sequencing that circumvents reverse transcription or amplification steps.
LUISA - Low Cost Unit for Sequencing Applications
University of São Paulo
Over the past few years, next generation sequencing with short and long reads, such as those generated by Oxford Nanopore Technologies platforms, have had a revolutionary advance for molecular biology, and made microbial genome sequencing cheap and accessible. However, handling long-reads and its high error rate poses a challenge which involves high computational and financial costs. In this regard, we developed LUISA, an embedded hardware with Ubuntu OS, several bioinformatic tools and three hardware options, GPU- and CPU-based (eGPU process unit) that can be powered by solar energy. Our preliminary benchmark indicates that LUISA can be at least three times faster than CPU-based Systems. LUISA will benefit to genomic surveillance and epidemiological studies of endemic diseases for low income countries, providing a cost-effective open solution option, making nanopore sequencing analysis simple, feasible and democratic for low income countries.
Dr. Cerdeira gained a B.Sc. in Computer Science, M.Sc. in Bioinformatics and a Ph.D. in Sciences at University of São Paulo, with an internship period at the University of Melbourne with Dr. Kathryn Holt. She is currently a research fellow in bioinformatics at University of São Paulo, Biomedical Sciences Institute in the Microbiology Department. Dr. Cerdeira has more than 8 years’ experience in bioinformatics and genomic analyses of large-scale sequencing data from animal, environmental and clinical sources, as well as knowledge of electronics, infectious diseases and molecular evolution. She has developed several bioinformatic pipelines using short- and long-reads for tracking outbreak dissemination of pathogenic bacteria (ESKAPE) using the One Health approach for evaluating antimicrobial drug resistance profiles; virulome, plasmidome and pangenome content, and phylogenetic analysis to discover transmission routes in South America. Additionally, she has developed and is still working on the development of several low-cost hardware units, such as the LUISA project, and pipeline and GPU-based tools that could help reduce the data processing time, providing feasible and democratic nanopore sequencing analysis for low income countries.
A new dawn in research and career progression
Plateau State University, Bokkos
The advent of nanopore technology holds lots of promise for resource limited communities. The application of molecular techniques to answer biological questions is still in its formative stage in Nigeria. The lack of electricity and other research basics makes nanopore technology a viable option in settings like ours. Since the arrival of nanopore technology to our laboratory, it has been a dawn of a new era in research for us. We have successfully used nanopore to re-sequence the genome of Mucuna pruriens and have designed research that before now would only be possible in collaboration with other labs. Here, I present how the arrival of this technology has turned our research focus and design around.
Nnaemeka Nnadi is a lecturer at the Department of Microbiology, Plateau State University, Bokkos, Nigeria. He obtained his PhD in Medical Microbiology from Nnamdi Azikiwe University Awka. A budding molecular biologist, Dr. Nnadi focuses on using bioinformatics to study the population genomics, evolution and pathogenesis of pathogenic microorganisms in Nigeria, using limited resources as is the case in Nigeria to study gene functions and gene interactions.
Food & Drug Administration
Data for breakfast
Cold Spring Harbor Laboratory
Data for breakfast
Data for breakfast
Oxford Nanopore Technologies
Culture-free detection of boxwood blight to improve disease diagnosis and prevention
Virginia Tech, USA
Marcela Aguilera Flores
The Oxford Nanopore Technologies MinION sequencer offers the potential for cost-effective and fast identification of plant pathogens directly from metagenomic DNA of infected plants without pathogen isolation. The entire process, from sample preparation to bioinformatics analysis, can be done in almost any laboratory or even in the field. Here, we used the MinION for the detection of boxwood blight, which is a recently emerged disease of the ornamental bush boxwood. The disease is caused by the fungus Calonectria pseudonaviculata (Cps) and can lead to complete defoliation and dieback. Conventional diagnosis relies on microscopic imaging of Cps spores after days, or even weeks, of incubation. However, since contact is the main route for disease propagation and infected plants are initially asymptomatic, early diagnosis and quarantine are thus necessary to prevent spread of this disease and reduce the damage it causes. After sequencing metagenomic DNA extracted from infected and healthy boxwood, we used BLASTN and MetaMaps with a custom database of Cps and other plant-pathogenic fungi to attempt detection of Cps. Although in small quantities, we were able to confirm with both software the presence of Cps in samples at all levels of infection, with the severe samples containing the highest number of sequences identified as Cps. Improvements to DNA extraction protocols and bioinformatics algorithms will be required to detect Cps in greater quantities in mildly symptomatic or even asymptomatic plants.
Highly multiplexed single-cell full-length cDNA sequencing with 10x Genomics and R2C2
University of California, Santa Cruz
The vast majority of single-cell studies have used Illumina’s short-read technology, which is state-of-the-art for quantifying gene expression but unable to accurately quantify RNA isoform expression. However, long-read technology excels at isoform sequencing because it preserves exon connectivity. Although long-read sequencing holds great promise for the characterization of single cells, studies thus far have been restricted to a handful of cells (~100) or low coverage per cell (~250 reads/cell). To overcome these limitations, we used the 10x Genomics platform in conjunction with R2C2 nanopore sequencing to perform highly multiplexed, single-cell, full-length cDNA sequencing on 3,000 peripheral blood mononuclear cells (PBMCs). Using the PromethION and MinION, we generated 18 million consensus reads with 96% base accuracy and were able to demultiplex 70% of them into individual cells. With a median of 5,000 reads per cell, we were also able to reproduce cell clusters based on gene expression data produced from short reads as well as identify differential isoform expression between single cells and cell types. Additionally, our 10x-R2C2 data enabled us to characterize variations in antibody isotypes, TCR isoforms, and antibody light chains. By uniting technologies to perform long, highly accurate, and single cell nanopore sequencing, our study brings forth a more powerful method for studying PMBCs and transcriptomics as a whole.
Single-molecule long-read sequencing reveals the chromatin basis of gene expression
The Ohio State University
Genome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remains poorly studied at the single-cell level. Here, we present a new experimental approach MeSMLR-seq (methyltransferase treatment followed by single-molecule long-read sequencing) for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules, and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. MeSMLR-seq offers direct measurements of both nucleosome-occupied and nucleosome-evicted regions on a single DNA molecule, which is challenging for many existing methods. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding the transcription start site for silently and actively transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin status spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming. Especially, we revealed the coupled changes of chromatin accessibility for two neighboring glucose transporter genes in response to the change of glucose concentration.
Deployment, training and sustainment of NGS in low-resource settings — case studies from Nigeria and Papua New Guinea
Biothreat detection capabilities are most critically needed in under resourced remote settings and border regions. In the past six months, GSSHealth has deployed MinION to remote sites in Nigeria and Papua New Guinea with the goal of identifying gaps and opportunities for the field deployment and sustainment of NGS as a biothreat detection tool. In both countries, partner organizations were provided local training, equipment, reagents and remote support to examine environmental samples in selected locations and are now being helped to develop additional research projects using the technology. In support of fully off the grid data acquisition, battery operated solutions were deployed for nucleic acid extraction and quantification, as well as library preparation and sequencing. Field data analysis was limited by requirements for internet connectivity to run ARMA & WIMP analysis and instead, a combination of offline and off-site solutions were tested. We will present on the challenges and barriers to the use of NGS in low resource field settings examining training requirements for local personnel, logistical challenges, community concerns, local data analysis and sustainability. This information, the algorithms developed for site and sample selection, baseline monitoring, and detection of potential biothreat signatures will inform future work on deploying dispersed surveillance networks to facilitate early detection of disease agents and emergency response in outbreak situations.
Nanopore sequencing of novel highly pathogenic avian influenza viruses from clinical samples: rapid pathotyping, full genomes, and novel defective interfering viral RNA
University of Alaska Anchorage
Highly pathogenic avian influenza viruses (HPAIV) cause mortality in wild birds and poultry, characterized by multi-organ inflammatory disease and neurological signs. In humans, HPAIV induced hypercytokinemia, pulmonary infiltrates and lung cell death. In order to better understand HPAIV genotypes and virus-host interactions, we adapted Oxford Nanopore MinION sequencing technology for whole genome sequencing of viruses from wild birds and domestic poultry, with a focus on samples collected in migratory wetlands and nearby poultry in Ukraine. HPAIV genomes were amplified from clinical tissue samples by multi-segment RT-PCR and cDNA were sequenced on a MinION Mk1B device using ligation sequencing protocols (LSK108/109). Using an iterative reference-based approach for genome assembly, H5N8 (clade 184.108.40.206B) and locally reassorted H5N5 HPAIV were readily distinguishable from low pathogenic H5 viruses, highlighting the utility of nanopore sequencing on the MinION platform for rapid pathotyping of HPAIV from outbreaks. Long read nanopore sequencing also mapped defective interfering (D.I.) RNA, an immunostimulatory influenza replication product, in clinical samples of HPAIV infection of wild mute swans. Junction sites in D.I. RNA suggest a structural model where these aberrant RNA species are produced by interaction of antiviral host factors with the viral RNA-dependent RNA polymerase.
Single-molecule, full-length transcript sequencing reveals disease-associated isoforms
Alternative splicing generates differing RNA isoforms that govern the phenotypic complexity of eukaryotes and its malfunction underlies many diseases, including cancer and cardiovascular disease. However, there are few methods to quantify transcript isoforms in cells, which is needed to elucidate their role in health and disease. Here, we used human-induced pluripotent stem-cell derived cardiomyocytes (iPSC-CMs) as a model to establish an experimental and analytical pipeline that accurately quantifies full-length transcriptome based on 21 million high-quality Oxford Nanopore Technologies sequencing reads. Our method overcomes the high false positive rates caused by sequencing artefacts in full-length transcript identification, achieving 97.6% identification accuracy estimated by synthetic isoform controls. We generated a quantitative full-length isoform annotation with 36,765 transcript isoforms in 11,707 genes, comprising of 24.1% of protein-coding isoforms in the GENCODE comprehensive annotation. Using our iPSC-CM model, we performed differential expression analysis on full-length transcriptome in the presence of an RBM20 mutation associated with aggressive dilated cardiomyopathy (DCM), which is caused by defective splicing. This revealed 38 transcript isoforms of 34 genes that are mis-spliced in the mutant. Notably, while the overall expression level of nine of these genes was unchanged in the mutant, our approach revealed significant differences in expression of specific isoforms, demonstrating the need to quantify full-length isoforms in gene expression analyses. We applied the same approach to study LMNA mutations in DCM patients, whose effects were previously unresolved by short-read RNA-seq. Our differential isoform analysis enabled us to reveal the detailed noncanonical splicing events caused by a splice-site mutation, leading to nonsense-mediated decay of LMNA transcripts. Our findings demonstrate that splicing is precisely regulated at the isoform level, and thus full-length isoform quantification instead of gene-level quantification is required in gene expression studies.
Using SIP and Oxford Nanopore MinION sequencing to uncover active bacterial and eukaryotic microbial communities in blueberry farm and forest soil systems
Highbush Blueberry (Vaccinium corymbosum L.) is a long-lived woody perennial that grows in unique (acidic, sandy, nutrient poor) soils. Although blueberry fields can remain productive for decades, older fields decline in overall plant health and productivity. To begin addressing soil health, we characterized the active members of the rhizosphere microbiome by stable isotope probing (SIP) and rRNA operon profiling. Over 24M raw reads were obtained for eukaryotic rRNA operons, basecalled using Guppy 3.2.2., and screened by Discontinuous MegaBlast against the Unite ITS database. Preliminary results indicate 1) DNA synthesis from 13C-15N Bioexpress is dominated by 3 eukaryotic fungi - Banoa ogasawarensis, Acidea extrema, and Acidomyces sp.; and 2). Declining soil demonstrated 2 to 400—fold more active Mucor moelleri, Mortierella elongata, Mortierella rishikesha, and Mortierella chlamydospora than forest soil or “good” farm soil. The active bacteria were mainly Firmicutes, Proteobacteria, and Actinobacteria, including the classes Bacilli (n=385 OTUs), Betaproteobacteria (n=298 OTUs), Gammaproteobacteria (n=98 OTUs), Actinobacteria (n=266 OTUs). There was no major difference in active bacteria for the top 10 species of the Betaproteobacteria or the Bacilli. Only slight differences between “good” and “declining” soil could be observed in the active Actinobacteria.
Human spaceflight induces telomere elongation and diplotype-resolved repeats
Weill Cornell Medicine
Telomeres are the natural ends of linear chromosomes that shorten with cell division and with aging. Longitudinal analyses of telomere lengths can provide an integrative and informative biomarker of general health and aging. Here, we assessed telomere length dynamics using the PromethION Oxford Nanopore sequencer for whole-genome sequencing (WGS) in four groups of samples: (1) identical twin astronauts and unrelated astronauts during one year and six month missions onboard the International Space Station (ISS), (2) all Genome in a Bottle (GIAB) human genome standards from NIST, (3) irradiated vs. control Plasmodium falciparum specimens, and (4) newborns vs. centenarians. Chromosome aberrations were also analyzed to evaluate telomere-related instability (e.g., fusions), as well as to assess DNA damage responses (DDRs) to ionizing radiation (IR) exposure during spaceflight, such as galactic cosmic ray (GCR)-induced damage. Telomere lengths were scored in a double-blind protocol and validated with qPCR, Telo-FISH, and HiFi PacBio reads. Across all experimental samples, we observed significantly longer telomeres (9.1x10-8) after exposure to ionizing radiation, including during spaceflight for 6-month and 1-year astronaut missions, as well as for P. falciparum using GCR-based IR. The GIAB samples did not exhibit longer telomeres after passage in culture, but served as useful controls, and the centenarians confirmed shorter telomeres as a function of time. For the astronauts, telomere length shortened rapidly upon return to Earth, and also exhibited more shortened telomeres after spaceflight exposure. Also consistent across these astronauts was evidence of chromosomal and telomeric DNA damage during spaceflight, with novel appearance in non-canonical (non-TTAGGG) telomeric repeats and DNA loops, indicating unique forms of structural variation that can only be resolved with long reads or structural mapping probes. Finally, we report an open-source suite of tools for mapping and analyzing these data called EdgeCase, which plots variation across both haplotypes (diplotype) at once.
Species-level evaluation of the human respiratory microbiome
Changes to human respiratory tract microbiome may contribute significantly to the progression of respiratory diseases. However, there are few studies examining the relative abundance of microbial communities at the species level along the human respiratory tract. Bronchoalveolar lavage (BAL), throat swab, mouth rinse, and nasal swab samples were collected from 5 subjects. Bacterial ribosomal operons were sequenced using the Oxford Nanopore MinION to determine the relative abundance of bacterial species in 4 compartments along the respiratory tract. Over 1.8 million raw operon reads were obtained from the subjects with ~600K rRNA reads passing QA/QC (70-95% identify; >1200 bp alignment) by Discontinuous MegaBlast against the EZ BioCloud 16S rRNA gene database. Nearly 3600 bacterial species were detected overall (> 750 bacterial species within the 5 dominant phyla: Firmucutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria. The relative abundance of bacterial species along the respiratory tract indicated most microbes (95%) were being passively transported from outside into the lung. However, a small percentage (<5%) of bacterial species were at higher abundance within the lavage samples. The most abundant lung-enriched bacterial species were Veillonella dispar and Veillonella atypica while the most abundant mouth-associated bacterial species were Streptococcus infantis and Streptococcus mitis. Most bacteria detected in lower respiratory samples do not seem to colonize the lung, however over 100 bacterial species were found to be enriched in bronchial lavage samples (compared to mouth/nose) and may play a significant role in lung health.
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
|Hotel||Walk from venue||Standard rate|
|Doubletree by Hilton NYC Chelsea||15 min||$359|
|Hyatt Herald Square||16 min||$389|
|Holiday Inn Manhattan 6th Ave - Chelsea||10 min||$329|
|Martinique New York on Broadway - Curio Collection by Hilton||16 min||$359|