Participants of this masterclass will learn about:
· Considerations when beginning your experimental planning
· Leveraging the Nanopore Community resources for guidance on various aspects of your experimental design
· How to approach experiments across a range of applications
Participants of this masterclass will learn about:
· The importance of extracting high-quality nucleic acids to get the most from your nanopore sequencing experiment
· How to optimise extraction methods to suit your experimental goals — including generating ultra-high molecular weight DNA
· How to check the quality of your extracted DNA and RNA
Participants of this masterclass will learn about:
· The range of nanopore sequencing kits available, including options for DNA, RNA, and ultra-long reads
· How to select a nanopore sequencing kit to suit your experimental goals
· The resources available online to help guide you through selecting a nanopore sequencing kit for your application
Participants of this masterclass will:
· Learn how to load a sequencing library on to a Flongle Flow Cell
· Have the chance to practice flow cell loading during the masterclass, using their own demo kit
Viewers of this Showcase demonstration will:
Participants of this masterclass will learn about:
· The different file types and formats involved in nanopore sequence data analysis
· The command-line and its importance
· Foundational analysis principles, focusing on data QC and alignment
Participants of this masterclass will learn about:
· The nanopore secondary analysis solutions available for different applications
· Representative analysis workflows, including for genome assembly, detection of structural variants, and methylation
· Additional resources, including how to explore nanopore sequence data using Oxford Nanopore open datasets
Gordon Sanghera was co-founder of Oxford Nanopore, together with Hagan Bayley and IP Group, and was appointed CEO in June 2005. He brings over 20 years' experience in the design, development, and global launch of disruptive platform sensor technologies. Dr. Sanghera’s Ph.D. in bioelectronics sensing was followed by a career at MediSense, an Oxford spin-out that delivered a new generation glucose technology to the market. Following the acquisition of MediSense by Abbott Laboratories, Dr. Sanghera held both UK and US VP and Director-level positions, including VP Worldwide Marketing, Research Director, and Manufacturing Process Development Director. Before its acquisition by Abbott, Gordon led the R&D of Medisense Inc. where he was instrumental in the launch of several generations of blood glucose bio-electronic systems for the consumer and hospital medical markets. He has also developed and validated production processes to meet with the regulatory requirements for USA and Europe. Gordon has a Ph.D. in bio-electronic technology and a degree in Chemistry.
Gordon Sanghera
Brain tumours cause the most cancer-related deaths in children while five-year survival in adults is approximately 20%. The first line of treatment for these patients is surgery to debulk the tumour, but gross total tumour resection is not always possible because of location, age, comorbidities etc., and the extent of resection depends mainly on tumour subgroup. However, precise diagnosis is typically uncertain at surgery. Using nanopore sequencing and DNA methylation classification, we could give the neurosurgeon the exact tumour subgroup within 100 minutes from sample collection. This would have influenced surgical strategy in 12 out of 20 patients evaluated perioperatively.
Dr. Luna Djirackor is a post-doctoral fellow at the Oslo University Hospital in Norway. She has a keen interest in cancer diagnostics. Her Ph.D. at the University of Liverpool, UK, focused on the characterization of stem cells in uveal melanoma. This showed her the translational possibilities of molecular diagnostics. Now at the Institute for Surgical Research, together with the neurosurgical team, Luna has used nanopore sequencing to classify brain tumours and influence neurosurgery in real time.
Luna Djirackor
We applied long-read and short-read RNA sequencing to identify full-length transcripts and quantify their expression during lineage commitment of human neuroblastoma cells. We compared the output of the technologies and confirmed the sensitivity of Oxford Nanopore long reads in quantifying gene and transcript expression and its power for identifying differential expression. Not only did we identify 5,656 differentially expressed transcripts, but we also report differential transcript expression for loci not differentially expressed at the gene level, highlighting the importance of investigating transcripts instead of genes. We also identified cases of isoform switching within the same gene during cell differentiation. We highlight the importance of using long-read sequencing to call expression and usage at the transcript level in order to understand the mechanisms enacting cellular differentiation.
Wilfried Haerty completed his Ph.D. in 2004 at the University Paris VI, followed by postdocs at McMaster University and University of Oxford in 2011, investigating splicing regulation and the evolution of coding and non-coding genes. Since joining the Earlham Institute in late 2015, his group has been investigating splicing diversity at different scales, single cells, cell cultures, and tissues. We analyse long-read data to annotate novel features and transcripts, assess their regulation during differentiation and development, aiming to identify potential therapeutic targets.
Wilfried Haerty
Epigenetics & human disease
Structural variation (SV) is the largest divergence of nucleic acids across human genomes and are strongly associated with human disease, evolution, gene regulation, and other phenotypes. Recently, long-read sequencing technologies have provided the opportunity to comprehensively identify SVs at high resolution. However, due to high sequencing errors and the complexity of SVs, there remains technical issues, even for the state-of-the-art SV calling approaches. Hence, we propose cuteSV, a sensitive, fast, and scalable alignment-based SV detection approach to comprehensively discover diverse SVs with outstanding performance, even with low coverage datasets. Benchmarking results indicate cuteSV is suitable for large-scale genome projects, due to its excellent SV yields and ultra-fast speed. Here, I present the overall framework and algorithm and provide a detailed outline for researchers to use cuteSV correctly and comprehensively.
Tao Jiang is a lecturer at the Center for Bioinformatics at Harbin Institute of Technology in China. His research focuses on developing methods for detecting the full spectrum of genomic structural variations and integrating these genetic resources into relevant, cutting-edge research. With the aid of long-read sequencing technologies, he has developed several tools for characterizing structural variation in the human genome, such as cuteSV and rMETL. Tao Jiang recently participated in the 100,000 Genomes project in China where he designed the overall technical route, including sample whole-genome sequencing and alignment, cohort-based joint-variant calling, large-scale functional annotation, and population genetic analysis.
Tao Jiang
Rapid improvement in Oxford Nanopore sequencing technology is enabling highly accurate genome inference. In collaboration with the genomics group at Google Health, we have developed a long-read small variant calling pipeline that produces state-of-the-art results for Oxford Nanopore reads (Shafin et al. 2021 biorxiv). Our nanopore-based method outperforms the short-read-based single nucleotide variant identification method at the whole-genome scale and produces high-quality single nucleotide variants in segmental duplications and low-mappability regions where short-read-based genotyping fails. In collaboration with Chan Zuckerberg Initiative (CZI), we developed Shasta, a de novo assembler, and demonstrated the ability to sequence and assemble eleven human genomes in nine days with a single PromethION device (Shafin et al. 2020 Nature Biotechnology). We have improved the existing de novo assembler, Shasta, to achieve higher contiguity and accuracy. In this talk, I will discuss the variant calling pipeline PEPPER-Margin-DeepVariant and the improvements in the Shasta de novo assembly methods that enable accurate genome inference with nanopore reads.
Kishwar Shafin is a graduate student at UC Santa Cruz Genomics Institute. His research is focused on developing methods based on deep neural networks for accurate human genome inference. He is a member of the group that showed sequencing and de novo assembly of eleven human genomes in nine days on a PromethION device (Shafin et al. 2020 Nature Biotechnology). More recently, he was involved in the nanopore category winning submissions at PrecisionFDA truth challenge v2, demonstrating the first nanopore-based method comparable with short-read-based single nucleotide variant identification methods (Shafin et al. 2021 biorxiv).
Kishwar Shafin
Dan Turner is Vice President, Applications at Oxford Nanopore Technologies. He provides leadership for multi-disciplinary teams in Oxford, New York, and San Francisco. The Applications group aims to bring together sample prep technologies, genomics applications, and bioinformatics, to expand the utility of Oxford Nanopore Technologies devices and illustrate the benefits of these technologies to the wider world. Before joining Oxford Nanopore Technologies, Dan was Head of Sequencing Technology Development at the Wellcome Trust Sanger Institute, and prior to this he held postdoctoral positions at the Sanger Institute and Cornell University Medical College in Manhattan.
Dan Turner
Sonic Hedgehog medulloblastoma (SHH-MB) is a malignant childhood brain tumor. In this study, we analyzed a single case of SHH-MB with a germline TP53 mutation (Li-Fraumeni syndrome) using a combination of short-read and long-read sequencing. The sample is characterized by complex inter- and intra-chromosomal genomic rearrangements indicative of chromothripsis, as well as disruption of DNA methylation levels. Nanopore sequencing enabled reconstruction of the genome and facilitated investigation of the interplay between the genomic and epigenetic effects.
Rene Snajder is a Ph.D. student at the German Cancer Research Center in Heidelberg, via the Helmholtz Information & Data Science School for Health. With a background in computer science, medical informatics, and software engineering, Rene has been working on machine learning problems involved in the integration of genetic and epigenetic modalities, both in long- and short-read sequencing data.
Rene Snajder
Rare diseases collectively affect millions of individuals worldwide. Although the majority are suspected to be genetic in origin, the molecular basis of many rare diseases is unknown. Here we discuss our experience using Oxford Nanopore Technologies’ long-read genome sequencing to characterize structural variation in individuals with known or suspected genetic syndromes. Nanopore sequencing was sensitive to the detection of pathogenic structural variants, allowing phasing of private, recurrent, and founder variants. Our findings helped inform the interpretation of candidate causal variants and together help establish a foundation for the clinical application of long-read genome sequencing as a comprehensive test for rare genetic diseases.
Katherine Dixon recently completed her Ph.D. in Medical Genetics at the University of British Columbia and is currently a Postdoctoral Fellow at BC Cancer in Vancouver, Canada. Using genome and transcriptome sequencing, Katherine studies the molecular basis of rare diseases, including cancer predisposition syndromes and congenital disorders.
Katherine Dixon
Zoe McDougall, VP of Corporate and Communications, joined Oxford Nanopore in 2008. As part of the leadership team, she works across corporate and commercial teams at the Company. Zoe started her career in sales & marketing at SmithKline Beecham. She subsequently managed a range of strategic scientific and healthcare communications at the agency Porter Novelli, where clients included GSK, BMS, Pfizer and a range of other healthcare clients. During this time Zoe also worked on crisis communications around the nvCJD issue. Before joining Oxford Nanopore, Zoe was part of the IPO team and subsequently led investor relations for a UK specialty pharma company Sinclair Pharma. In 2005 she worked with a medical humanitarian organisation in Sri Lanka.
Zoe McDougall
Human papillomavirus (HPV) is a necessary but insufficient driver of cervical cancer. We used nanopore sequencing to generate genome, methylome, and transcriptome data for cervical cancers from Uganda (HTMCP) and the USA (TCGA). HPV integration events underlie complex intra- and inter-chromosomal structural variants that sometimes resulted in highly expressed gene fusions. Haplotype-resolved DNA methylation showed human enhancer dysregulation affecting the HPV-containing allele and allowed us to determine the methylation status of individual viral integration events. Long-read profiling will thus reveal new cancer features that were previously cryptic, and such features will clearly advance cervical cancer biology and perhaps cancer genomic medicine.
Vanessa Porter is a Ph.D. candidate in Dr. Marco Marra’s lab at Canada’s Michael Smith Genome Sciences Centre in Vancouver. This is Vanessa’s fourth year in the Doctor of Medical Genetics program at the University of British Columbia. Her project investigates how HPV in different contexts affects the structure and regulation of cervical cancer genomes using multi-omics analyses.
Vanessa Porter
To understand the mechanisms underlying the convergent adaptation to myrmecophagy (the consumption of ants and/or termites) in mammals, the ConvergeAnt project proposes an integrative approach combining morphology, genomics, and metagenomics. The metagenomics part of the project focuses on the role of the gut microbiome in facilitating the digestion of the large amount of ants and termites ingested. We aim to compare the gut microbiomes of convergent myrmecophagous species, both in terms of taxonomic and functional gene content by identifying bacterial taxa, reconstructing their genomes, and analysing chitin degradation pathways. Using the MinION portable device, we sequenced metagenomes from 12 field-collected faecal samples of three myrmecophagous species: the aardvark (Orycteropus afer), the southern aardwolf (Proteles cristatus), and the ground pangolin (Smutsia temminckii). We did taxonomic profiling of those metagenomes, assembled them using three long-read metagenomic assemblers, and extracted potentially relevant metagenome-assembled genomes (MAGs) to use them to investigate functions possibly linked to chitin degradation.
Sophie Teullet received her Master’s degree in Ecology and Evolution from the University Paul Sabatier in Toulouse, France, last year and is currently a Ph.D. student at the University of Montpellier in the Institut des Sciences de l’Evolution of Montpellier (ISEM), France. As part of the ConvergeAnt project, her Ph.D. project focuses on dietary adaptations in the genomes and microbiomes of ant-eating mammals.
Sophie Teullet
Elizabeth Skippington is a scientist in the Department of OMNI Bioinformatics at Genentech, South San Francisco, USA. She is a graduate of the University of Queensland in Genomics, and completed her postdoctoral training at Indiana University. A primary focus of her research is the investigation of antibiotic resistance mechanisms using DNA sequencing technologies.
Elizabeth Skippington
Across the world, wild populations are declining at an unprecedented rate. Environmental DNA (eDNA) research can be leveraged to quantitatively monitor intra- and inter-specific diversity and evaluate the resilience of the world’s ecosystems in a scalable and non-invasive manner. We used eDNA to monitor the critically endangered kākāpō parrot (Strigops habroptilus) and leveraged adaptive sampling to correctly predict the presence of an individual kākāpō. This pilot study is a first step towards expanding the application of eDNA research to inferring fitness-related parameters, such as genetic structure and inbreeding, with important implications for in-depth monitoring of rare and elusive species.
Lara’s research focuses on how genomics research can benefit and be incorporated into the conservation of nature. During her Ph.D. at the University of Cambridge and EMBL-EBI, she applied and developed methodology in the fields of statistical genomics, single-cell genetics, and metagenomics. As a Research Fellow at the University of Otago, Lara now leverages this expertise in genomics to support the conservation of critically endangered species endemic to New Zealand, and to benchmark environmental DNA approaches for biodiversity monitoring.
Lara Urban
We sequenced the genomes of 14 Arabidopsis thaliana GABI-Kat T-DNA insertion lines and detected complex T-DNA insertion arrays and 11 previously unknown T-DNA insertion sites. Chromosome fusions and translocations were identified as by-products of T-DNA insertion mutagenesis, including compensating translocations without T-DNA at the junctions. We developed an automated workflow which supports the in-depth analysis of long-read data from T-DNA insertion lines. The workflow from DNA extraction to (local) assembly of the T-DNA loci can easily be completed within a week, offering fast access to a complete line characterisation, including previously undetected insertions.
During his time at the CeBiTec at Bielefeld University, Boas Pucker sequenced the genomes of several plants including yam, sugar beet, and grapevine. Now, he is working as a DFG Research Fellow on the evolution of the betalain biosynthesis in the Caryophyllales at the University of Cambridge.
Boas Pucker
Mutations in RNA splicing factors are commonly observed in cancer. For example, splicing factor 3B subunit 1 (SF3B1) mutations are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and are also associated with a high-risk of leukemic transformation in clonal hematopoiesis (CH) individuals. SF3B1 mutations are associated with aberrant RNA splicing, resulting in increased cryptic 3’ splice site usage, and often lead to refractory anemia with ringed sideroblasts clinical phenotype. However, the full impact of SF3B1 mutations on cellular fitness leading to clonal outgrowth in humans remains largely unknown. The study of SF3B1-mediated splicing aberrations has been hampered by the inability to distinguish mutated and wildtype cells in human MDS and CH samples on the one hand, and the limitation of short-read single-cell RNA sequencing to adequately cover splice junctions on the other hand. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT) with long-read full-length cDNA profiles at single-cell resolution, using Oxford Nanopore sequencing with the PromethION. This allowed for the simultaneous profiling of gene expression, somatic mutation status, and alternative splicing in single cells. Furthermore, we significantly improved read recovery previously reported with Oxford Nanopore flow cells. Using a biotin enrichment protocol, GoT-Splice allowed for the selective amplification of full-length reads with the accurate structure, thus maximizing reads that can be used for confident alternative splice junction calling at the single-cell level. We applied GoT-Splice to CD34+ cells from MDS patients with SF3B1mut to study how SF3B1 mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant vs. normal hematopoietic progenitors revealed an increasing fitness advantage of SF3B1mut cells with myeloid differentiation, resulting in a build-up of SF3B1mut cells in the erythroid progenitor state. GoT-Splice uniquely allowed us to jointly capture SF3B1 mutations and genome-wide splicing information to chart the variation of splicing alterations in SF3B1mut cells within individual hematopoietic cell types. We not only showed increased detection of cryptic 3’ splice signal over previous bulk RNA-sequencing methods but also revealed that MDS patients exhibit distinct cryptic 3’ splice site usage in SF3B1mut cells as a function of hematopoietic progenitor cell identity. Specifically, we observed the largest increase in cryptic 3’ splice site usage in the erythroid progenitors, consistent with the ineffective erythropoiesis phenotype associated with this mutation. Similar to MDS, the application of GoT-splice to CD34+ progenitor cells from individuals with SF3B1mut CH revealed increased mutant cell frequency in EPs and cell-type specific cryptic 3’ splice site usage in SF3B1mut cells. In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of somatic splicing factor mutations on cellular fitness and its molecular determinants from the earliest phases of clonal blood disorders (CH) to overt MDS, providing a widely applicable methodology and related analytics to study the effect of somatic mutations on isoform-level gene expression patterns in single cells directly in human samples.
Dan A. Landau, MD, Ph.D., is a Core Faculty Member at the New York Genome Center. He holds a joint appointment as Associate Professor of Medicine in the Division of Hematology and Medical Oncology and the Department of Physiology and Biophysics at Weill Cornell Medicine.
Dan A. Landau
Nick is Professor of Microbial Genomics and Bioinformatics in the Institute of Microbiology and Infection at the University of Birmingham and a Fellow at the Alan Turing Institute. He is supported by a Fellowship in Microbial Genomics Bioinformatics as part of the MRC CLIMB project. His research explores the use of cutting-edge genomics and metagenomics approaches to the diagnosis, treatment, and surveillance of infectious disease. Nick has so far used high-throughput sequencing to investigate outbreaks of important Gram-negative multi-drug resistant pathogens, and recently helped establish real-time genomic surveillance of Ebola in Guinea and Zika in Brazil. His current work focuses on the development and evaluation of novel molecular biology, sequencing, and bioinformatics methods to aid the interpretation of genome- and metagenome-scale data generated in clinical and public health microbiology.