Application of nanopore sequencing in clinical haematology
Blood cancers are together the 5th most common cancer and many patients are young adults. Besides, the most common cancers of childhood worldwide are acute lymphoblastic leukaemia and endemic Burkitt’s Lymphoma. Six of the 10 most lucrative cancer drugs are prescribed for haematological malignancies ($55.6billion by 2025). Their diagnosis requires a microscope and DNA-based precision diagnostics. But precision diagnostics is not always about identifying a simple base pair change, and requires the detection of all different types of mutations from one single (often small) patient sample. Examples of where DNA-based diagnostics are critical will be given and include all leukaemias and an increasing number of lymphomas. Their detection is recommended by the WHO classification of haematological malignancies. For example, the NHS test directory includes 177 different genetic aberrations.
The problem is that current multimodality testing in diagnostics laboratories is inadequate to deal with this demand on testing. Many of the conventional single gene assays lack sensitivity, speed and precision. Illumina whole genome sequencing (WGS) of tumour and paired germline has the potential to reveal all types of different mutations and global measures across the genome, but it is limited by the small fragment size and the need for large and expensive equipment. As part of the Genomics England Chronic Lymphocytic leukaemia Pilot have used information from Illumina WGS from 400 patients to develop an improved response prediction tool for chemoimmunotherapy that predicts patients who will be cured (manuscript in preparation). An alternative way specifically for diagnostics, is to combine targeted deep sequencing and error correction with shallow whole genome sequencing using the MinION. This method and variations of it can be applied globally in haematology. For example, the most common inherited anaemias, the haemoglobinopathies, are characterised by 1700 SNVs/indels and deletions across three genes. The most common of these, sickle cell disease, occurs in sub-Saharan Africa. About 10% of patients require confirmatory diagnosis by genetics. Life-saving therapies are available for this disease, and knowledge of the presence of the condition in the fetus could significantly streamline neonatal screening programmes around the world. We have clinically validated a proprietary method for non-invasive testing for sickle cell disease from maternal plasma (pre-published in BioRxiv) and have also developed a nanopore-based test for diagnosing haemoglobinopathies from germline DNA without the need for PCR amplification.
Finally, plasma-derived DNA can also be used in other clinical indications in sub-Saharan Africa. The most common childhood cancer in the region is endemic Burkitt’s lymphoma. This is caused by EBV infection in early childhood. With simple treatment, over 90% of patients can be cured. And treatment is free of charge in all African countries affected. Currently, over 90% of kids die. This is because children present late and are not diagnosed once in hospital because there is lack of trained surgeon and pathologists to establish the diagnosis from an invasive biopsy across the region. We are now clinically validating a non-invasive method to diagnose this type of lymphoma from the blood using a combination of tumour and virus sequencing.
In conclusion: haematological diseases have always spearheaded innovations and discoveries in medicine, in particular genetics. Precision medicine is a reality for an increasing number of patients with blood diseases from targeted small molecules in leukaemias and lymphomas to gene therapy in the inherited blood diseases. The next step is to leapfrog diagnostics technologies and to introduce these advances globally as expensive cancer therapies are coming off patent and are increasingly available and on the WHO list of essential medicines. Ultimately, this approach will achieve a huge impact for a large number of patients world-wide.
Professor Schuh completed academic and clinical haematology training in Oxford and in 2006, she was appointed clinical lead for haematology laboratories, including molecular diagnostics, and has also been the clinical lead for chronic lymphocytic leukaemia and other lymphoproliferative disorders for the NHS Thames Valley Cancer Network. Over the past twelve years she has led over 30 early and late phase clinical trials in leukaemia as a principle or national chief investigator. A number of these led to NICE approvals and have changed clinical practice for patients in the UK and worldwide. As a result, she was recently appointed as the Chair of Chronic Lymphocytic Leukaemia Research in the UK by the National Cancer Research Institute. In addition to other national and international roles, she has also chaired the UK CLL Forum since 2016 that promotes training and education, and she has led the UK's guidelines writing group for CLL Therapy on behalf of the British Society of Haematology. Her second research interest is with the development, evaluation and implementation of new technologies for Precision Diagnostics, especially genomics. Her group published the first ever longitudinal study of the changes in the genomic landscape of patients undergoing treatment for leukaemia. She is the lead for the Genomics England Clinical Interpretation Partnership for haematological malignancies. Professor Schuh has received grants from the NIHR, Wellcome Trust, Technology Strategy Board, Cancer Research UK and Bloodwise and she has authored or co-authored over 80 peer-reviewed publications in the last five years.
Discerning the origin of Epstein-Barr virus in patients using nanopore-derived DNA methylation signatures
Epstein-Barr virus (EBV) infects the vast majority of the human population. In rare instances EBV is involved in the generation of lymphomas and other malignancies. The presence of elevated EBV levels in blood is currently used in the clinical diagnosis and subclassification of infectious disease and lymphoma; yet when patients present with detectible EBV in blood, it is uncertain if this represents an active infection or an evolving malignancy. Epigenetic changes occur as an essential component of the EBV life cycle, controlling the virus’s ability to infect, establish a chronic (latent) infected population of cells, and later reactivate to produce infectious virions. We have found that tumor-derived EBV DNA displays specific DNA methylation signatures and EBV virion-derived infectious particles or lytically-active (virus-replicating) cells contain largely unmethylated EBV DNA. Importantly, tumor-specific EBV methylation patterns are maintained in circulating plasma (cell-free) DNA. We have employed nanopore sequencing to interpret EBV DNA methylation signatures in patients. Analysis of tumor and plasma-derived EBV methylomes from lymphoma patients and donors with infectious EBV revealed highly discernable DNA methylation signatures, with higher levels of methylation in primary EBV+ lymphoma cases. In addition, we have uncovered an unexpectedly high complexity of EBV methylation patterns among tumors and evidence that epigenetic differences correlate with viral gene expression. These data may predict responses to combined antiviral and immunotherapeutic strategies in cancer patients. In summary, our work aims to develop approaches that harness a patient’s EBV epigenetic signature with the aid of nanopore sequencing to rapidly discern benign versus cancerous clinical scenarios and help direct the use of current and novel therapies.
Christopher Oakes is an Assistant Professor in the Departments of Internal Medicine/Division of Hematology and Biomedical Informatics at The Ohio State University. He performed his graduate studies at McGill University in Canada and post-doctoral studies at the German Cancer Research Center in Heidelberg, Germany. His laboratory investigates epigenomic, genetic and other molecular features of a broad range of hematological malignancies, with a focus on chronic lymphocytic leukemia and other non-Hodgkin’s lymphomas and acute myeloid leukemia. He explores high-throughput epigenetic and molecular profiling data and combines these analyses with functional evaluation of key genes and molecular pathways. His laboratory is interested in the developmental origins of epigenetic programs in lymphoid and myeloid malignancies and aim to uncover the ontogeny of disease development. Current research focuses on investigating the role that perturbation of gene function plays in establishing aberrant global epigenetic states and landscapes. Beyond fundamental tumor biology, he aims to develop novel molecular diagnostics clinical for diagnosis, stratification and prediction of treatment responses, as well as the identification of novel therapeutic targets.
Update from Oxford Nanopore Technologies
Clive is Chief Technology Officer at Oxford Nanopore Technologies. On the Executive team, he is responsible for all of the Company’s product-development activities. Clive leads the specification and design of the Company’s nanopore-based sensing platform, including strand DNA/RNA sequencing and protein-sensing applications with a strong focus on scientific excellence and successful adoption by the scientific community.
Clive joined Oxford Nanopore Technologies from the Wellcome Trust Sanger Institute (Cambridge, UK) where he played a key role in the adoption and exploitation of next-generation DNA sequencing platforms. This involved helping to set up the world’s largest single installation of Illumina (formerly Solexa) Genome Analyzers in a production sequencing environment, initially used to pioneer the 1000 Genomes Project. From early 2003 he was Director of Computational Biology and IT at Solexa Ltd, where he was central to the development and commercialisation of the Genome Analyzer (GA). Solexa was sold to Illumina for $650m in early 2007 after the successful placement and adoption of 12 instruments. The Solexa technology, now commercialised by Illumina, is the market-leading DNA sequencing technology driving the renaissance in DNA-based discovery.
He has a strong background in computer science and genetics/molecular biology and manages interdisciplinary teams including mechanical engineering, electronics, physics, surface chemistry, electrophysiology, software engineering and applications (of the technology). Clive applies modern agile management techniques to the entire product-development lifecycle. Clive has also held various management and consulting positions at GlaxoWellcome, Oxford Glycosciences and other EU- and US-based organisations. He has worked at the interface between computing and science, ranging from genetics to proteomics. He holds degrees in Genetics and Computational Biology from the University of York.
The real Simon Pure
Dan Turner is Vice President of Applications at Oxford Nanopore Technologies and is a highly experienced scientist who has worked in the field of next-generation sequencing for the last 11 years. Dan provides scientific leadership for multi-disciplinary teams in Oxford, New York and San Francisco. The Applications group aims to bring together sample prep technologies, genomics applications and bioinformatics, to expand the utility of Oxford Nanopore Technologies devices and illustrate the benefits of these technologies to the wider world. Before joining Oxford Nanopore Technologies, Dan was Head of Sequencing Technology Development at the Wellcome Trust Sanger Institute, and prior to this he held postdoctoral positions at the Sanger Institute and Cornell University Medical College in Manhattan.
Accurate detection of m6A RNA modifications in native RNA sequences using third-generation sequencing
From the battery of over 170 known RNA modifications, more than 70 have already been linked to human diseases, including neurological disorders and cancer, highlighting their importance in proper cellular functioning. Unfortunately, the limited availability of antibodies and chemicals selective to RNA modifications has so far limited our transcriptome-wide view to only a handful of RNA modifications. Consequently, the abundance, location, and function of the majority of RNA modifications remains unknown. To overcome these limitations, we have employed direct RNA sequencing from Oxford Nanopore Technologies, which allows direct sequencing of native RNA molecules, without any further amplification or reverse transcription step, thus potentially allowing for direct detection of RNA modifications in the full-length RNA transcripts. Using this technology, we have trained an algorithm that allows for the detection m6A RNA modifications in a quantitative manner and with single nucleotide resolution, finding that we can detect m6A RNA modifications with an overall accuracy of 90%. We then validate our findings in vivo, showing that our methodology can detect m6A modifications in yeast. As a control, we show that these modifications are not predicted by our algorithm in Ime4 knockout strains, which lack m6A. Our results open new avenues to investigate the universe of RNA modifications in full-length transcripts, with single molecule resolution. The establishment of the Oxford Nanopore platform as a tool to map virtually any given modification will allow us to query the epitranscriptome in ways that, until now, had not been possible. Future work can expand to other modifications like 5-methylcytosine (m5C), as well as provide additional thresholds for controlling specificity and sensitivity.
Eva Maria Novoa obtained her BSc in Biochemistry in 2007 with Honours, followed by an MSc in Bioinformatics in 2009. Since then, she has conducted research across three continents, including the Institute for Research in Biomedicine (IRB Barcelona) in Spain, the Massachusetts Institute of Technology and the Broad Institute in the USA, and the Garvan Institute of Medical Research in Australia. During these years, she has generated a substantial research profile in the field of protein translation and post-transcriptional regulation, using a combination of molecular biology, biochemistry and bioinformatic approaches. Since 2018, she has been Group Leader at the Center for Genomic Regulation (CRG) in Spain, in a dual appointment with the Garvan Institute, where she leads a team of 8 people. Her current work is focused on deciphering the language of RNA modifications, and how its orchestration can regulate our cells in a space-, time- and signal-dependent manner. Eva has received fellowships from EMBO, HFSP, “LaCaixa” and the ARC, and her work has been awarded with the Fisher Scientific Prize for Young Researchers (2013) given by the Spanish Society of Molecular Biology and Biochemistry, and the Young Researcher Award (2016) given by the Catalan Society of Biology.
Cyclomics: ultra-sensitive nanopore sequencing of cell free tumor DNA
In many types of cancer, tumor cells shed small (~150bp) DNA molecules in the blood or other body fluids. Detecting mutations in the cell-free DNA (cfDNA) content of liquid biopsies in cancer patients thus offers a unique opportunity for non-invasive diagnostics for the purpose of e.g. treatment monitoring treatment response or detecting recurrent disease. Reliably detecting mutations from minute amounts of tumor derived cfDNA is, however, highly challenging. To address this, we developed CyclomicsSeq, a novel sequencing approach leveraging the long-read nanopore platform to achieve single molecule detection accuracy. We have demonstrated proof of concept on head and neck cancer (HNC) patient samples and found that CyclomicsSeq can detect mutations anywhere in the TP53 gene with single-molecule accuracy. CyclomicsSeq thus offers a reliable liquid biopsy diagnostic assay which can be cost-effectively implemented in routine clinical workflows.
Dr. Jeroen de Ridder is a Principal Investigator and Associate Professor at the Center for Molecular Medicine of the University Medical Center Utrecht, as well as a junior PI at the Oncode Institute. He runs a bioinformatics lab which aims to create and apply innovative data science methods to advance our understanding of disease biology. His research efforts are always inspired by a biological question and typically deal with big data, such as large-scale genomics and epigenomics datasets. As a result, much of the research floats on machine learning and data integration algorithms. Recently, Dr. de Ridder, along with Dr. Kloosterman and Dr. Marcozzi, founded a start-up company Cyclomics, which aims to provide ultra-sensitive sequencing of cell free tumor DNA.
Telomere-to-telomere assembly of a complete human X chromosome
Release of the first human genome assembly was a landmark achievement, and after nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has yet been finished end to end, and hundreds of gaps persist across the genome. These unresolved regions include segmental duplications, ribosomal rRNA gene arrays, and satellite arrays that harbor unexplored variation of unknown consequence. We aim to finish these remaining regions and generate the first truly complete assembly of a human genome.
Here we announce a whole-genome de novo assembly that surpasses the continuity of GRCh38, along with the first complete, telomere-to-telomere assembly of a human X chromosome. In total, we collected 40X coverage of ultra-long Oxford Nanopore sequencing for the CHM13hTERT cell line, including 44 Gb of sequence in reads >100 kb and a maximum read length exceeding 1 Mb. This unprecedented coverage of ultra-long reads enabled the resolution of most repeats in the genome, including large fractions of the centromeric satellite arrays and short arms of the acrocentrics. A de novo assembly combining this nanopore data with 70X of existing PacBio data achieved an NG50 contig size of 75 Mb (compared to 56 Mb for GRCh38), with some chromosomes broken only at the centromere. Using this assembly as a basis, we chose to manually finish the X chromosome. The few unresolved segmental duplications were assembled using ultra-long reads spanning the individual copies, and the ~2.3 Mbp X centromere was assembled by identifying unique variants within the array and using these to anchor overlapping ultra-long reads. These results demonstrate that it is now possible to finish entire human chromosomes without gaps, and our future work will focus on completing and validating the remainder of the genome.
Karen H. Miga, PhD, is an Assistant Research Scientist at UCSC. Dr. Miga’s research program combines innovative computational and experimental approaches to produce the high-resolution sequence maps of human centromeric and pericentromeric DNAs.
Identification of new somatic structural variants and cancer driver genes using long-read nanopore sequencing
Third generation DNA sequencing technologies have been transforming genome medicine and cancer research, producing evidences for structural variations (SV’s) being the common and major driver of complex diseases and tumorigenesis. By taking advantage of the un-parallelled power of long-read and high-throughput capability of the Oxford Nanopore PromethION platform, we investigated the role of SV’s in cancer development. We sequenced DNA obtained from colorectal cancer biopsy and corresponding normal tissue-samples of Han Chinese. Using a comprehensive SV-calling pipeline that consists of ngmlr-sniffle, dynamic filtering, database search and comparison, manual curation, and break point mapping, we obtained high quality SV call sets. By using PCA, population structure, and frequency spectrum analyses, we identified a set of SV’s that are tumor specific. In addition to somatic point mutations in mismatch repair genes that are well known for causing colorectal cancers, we observed complex somatic SV’s that show evidence of chromothriptic rearrangements, the hallmark of the late stage tumors, that were focally localized to a terminal region of a chromosome in colorectal cancer samples. One of the complex somatic rearrangements was linked to the amplification of the gene that is essential for DNA recombination. Furthermore, we also observed a direct link between the expansion of microsatellites and SV’s, suggesting the microsatellite instability might drive the formation of SV’s and cause genome instability in colorectal cancers. Collectively, our results present the power of the Oxford Nanopore PromethION platform for high resolution analysis of SV’s in the human genome, which can lead to a better understanding of the molecular, biochemical, and cellular events that govern tumor progression.
As Director of the Grandomics Genome Institute, Min works with a talented group of scientists and technologists who develop new genomic solutions to enhance the strengths of the Oxford Nanopore platform for genome science and genome medicine. His team integrates existing and new methods to create a comprehensive pipeline to produce complete animal and plant genomes with a minimum number of gaps. His team also studies the origin, mechanisms, and roles of SV’s in adaptive evolution, complex diseases, and tumorigenesis.
Revealing mRNA alternative splicing complexity in the human brain
Identifying the cellular pathways underlying psychiatric disorders has great potential to improve patient lives. Voltage-gated calcium channels (VGCCs), including CACNA1C, have been linked to the risk of bipolar disorder and schizophrenia, so are promising targets for new treatments. VGCCs are also important in the cardiovascular system, meaning treatments must be tissue-selective. We aim to identify brain-enriched CACNA1C isoforms as novel targets for new treatments. We used targeted long-range nanopore sequencing to characterise full length transcripts of CACNA1C in human brain and mouse brain, heart and aorta. In human we identified >250, and in mouse >190, mRNA isoforms, exons and splice junctions; including many predicted to modulate protein function. In mice, splicing was clearly tissue-specific, and we expect that this underlying principle of tissue-specific splicing will be conserved between mice and humans. Our data show that splicing of CACNA1C in human brain is far more diverse than is currently appreciated. This information will be critical to reveal pathophysiological mechanisms, and to identify brain-enriched VGCC isoforms that may be novel targets for new psychiatric treatments.
Nicola Hall is a postdoctoral researcher at the University of Oxford in Department of Psychiatry with the Tunbridge group. She is using her background in molecular biology and RNA sequencing to investigate gene expression in the human brain. Her current work focuses on alternative splicing of the calcium channel CACNA1C, implicated in schizophrenia and bipolar disorder. Nicola completed her PhD in 2017 at the University of Oxford in the Department of Biochemistry.
Retroviral invasion of the koala genome
Using direct RNA sequencing to detect RNA structures in transcriptomes
Studying how an RNA folds is crucial to understanding RNA function inside cells. RNA secondary structure mapping using second generation short-read sequencing such as Illumina, provides large scale structure information but lacks the connectivity between structures along a transcript. Here, we utilize direct RNA sequencing to detect structure modifications. Using machine learning models, we demonstrate that we can accurately detect RNA secondary structures and their dynamics on known RNAs. Structure probing of the human embryonic stem cell transcriptome captures structural features seen in other high throughput structure datasets and allows us to detect structural information in individual isoforms of a gene. We show that structural differences between isoforms are associated with differential translation efficiencies, further expanding our understanding of structure and function inside cells.
Yue Wan received her BSc in Cell Biology and Biochemistry from the University of California, San Diego and her PhD in Cancer Biology from Stanford University under the mentorship of Howard Y. Chang. During her PhD, she developed the first high-throughput method for probing RNA structures genome-wide. Yue is a recipient of the NSS-PhD scholarship from the Agency for Science, Technology and Research (A*STAR) in Singapore and is currently a Principle Investigator in the Genome Institute of Singapore. She is a Society in Science- Branco Weiss Fellow, EMBO Young Investigator and CIFAR-Azrieli Global Scholar, as well as a recipient of the Young Scientist Award and L’Oreal-UNESCO for Women in Science, Singapore National Fellowship. She is interested in studying functional RNA structures and understanding their roles in regulating cellular biology.