Cas9 targeted enrichment for nanopore profiling of methylation at known cancer drivers
CpG methylation in the mammalian genome is known to alter the binding of transcriptional regulatory factors, and through this activity mediate changes in chromatin structure and gene expression. We have previously shown the ability to call the methylation status of CpG sites using the signal from nanopore sequencing, and we are aiming to leverage this to profile the methylation status at high sequencing depth of genes known to be involved in tumorigenesis. These efforts have focused on using Cas9-enrichment, sidestepping the relatively high cost/bp of nanopore sequencing while retaining its exquisite sensitivity and long-reads. This allows us to profile signals of methylation over a long range on a single DNA molecule, and by comparing cells with different malignant potential, we can provide new insight into the dynamics of CpG methylation patterns. Specifically, we have targeted the promoter region of the human telomerase gene (hTERT), which is known to have altered methylation patterns that reflect the malignant potential of different cell line. This region is typically difficult to profile with bisulfite amplicon sequencing because of the repetitive and high CG density. Using thyroid cancer cell lines, we have sequenced this region, and identified both methylation level and individual single-read methylation patterns in the samples.
Timothy Gilpatrick is a current MD/PhD student at Johns Hopkins University working in the lab of Winston Timp to leverage sequencing technologies for insight into epigenetic states and transcriptional regulation. He received his BSc in Biochemistry from the University of Delaware, where he worked on characterizing the role of lipoprotein-associated enzymes in atherosclerosis. Prior to starting his graduate studies, he worked as a research fellow at the National Institutes of Health, where his work centered on understanding how microRNAs regulate epigenetic silencing machinery in mouse ES cells. He hopes to marry his interests in medicine and genomics to work in the development and application of sequencing-based diagnostic techniques. When not in lab, Timothy enjoys city biking, yoga, and music production.
Clive Brown: Plenary from London Calling 2018
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.
Haplotyping of key cardiac disease genes using long-read sequencing
Hypertrophic cardiomyopathy, characterized by an overgrowth of the left ventricular heart wall, affects 1:500 in the population and can lead to atrial fibrillation, heart failure, and sudden death. Causative genetic mutations identified in sequenced patients most often occur in one of two genes: MYH7 (myosin heavy chain 7) or MYBPC3 (cardiac myosin binding protein c), key components of the cardiac sarcomere. Here, we use targeted long-read sequencing to connect rare genetic variants in sarcomeric genes to novel RNA isoforms. We additionally use long-read sequencing to haplotype cardiac-disease associated genes to identify targets for precision medicine therapeutics. Targeted, multiplexed long-read sequencing of key cardiac disease genes presents an efficient strategy for defining precision medicine targets and providing evidence for disease-causing mutations in cardiovascular disease.
Alex is a PhD candidate in Euan Ashley's lab at Stanford University, studying hypertrophic cardiomyopathy. Her thesis research involves sequencing and haplotyping clinically relevant genes in cardiac disease and using that information to design allele-specific, precision therapeutics.
Dainis, A.M. Cardiovascular precision medicine in the genomics era. JACC: Basic to Translational Science, 3, 313-326 (2018)
Metagenomic nanopore sequencing: RNA viruses from lab to field
Emerging and re-emerging RNA viruses cause a significant global disease burden, ranging from mild febrile illness to haemorrhagic fevers. Rapid and unbiased identification methods, such as metagenomic MinION sequencing, are vital for the identification and characterisation of emerging pathogens for which little prior knowledge is available. Portable methodologies for field use are required during such outbreaks, especially when they occur in resource-limited settings. We have investigated a range of clinical samples using a Sequence Independent Single Primer Amplification approach and have demonstrated that metagenomic MinION sequencing can elucidate full viral genomes directly from clinical samples for Chikungunya, Dengue and Lassa virus; across clinically relevant range of viral titers. Following our results we mobilized a research team and deployed in Nigeria to test and investigate the establishment of field metagenomic sequencing using the MinION. Our pilot study was expedited and utilized to support the largest reported outbreak of Lassa fever (LASV) in Nigeria.
Liana is a PhD student based at Public Health England; her project is part of the NIHR Health Protection Research Unit in Emerging and Zoonotic infections, a collaboration between Public Health England and the University of Liverpool. Her work focuses on investigating the application of metagenomic sequencing methods to viral clinical samples. Liana is interested in field sequencing, and utilised the MinION for metagenomic sequencing in Nigeria during the recent Lassa fever outbreak.
Nanopore sequencing of cancer genomes
Cancer genome sequencing is taking central stage in oncology. Despite improvements in genomics technology, the detection of structural variants from short-read cancer genome sequencing still poses challenges, particularly for complex variation. I will highlight our work on the generation of long-read sequencing data for cancer genomes and the extraction of high-quality sets of somatic structural variations from these data. I will further discuss experiments on the use of somatic breakpoints and mutations to track cancer in liquid biopsies using nanopore sequencing
Dr. Wigard Kloosterman is Group Leader and Associate Professor at the Department of Genetics within the Center for Molecular Medicine at the University Medical Center Utrecht in The Netherlands. The Kloosterman group has strong expertise in cancer genomics, bioinformatics and nanopore sequencing technology. Dr Kloosterman received a PhD from Utrecht University.
Native RNA sequencing of human polyadenylated transcripts
On behalf of the Nanopore RNA Consortium (Akeson, Brooks, Loman, Loose, Paten, Simpson, Timp, and Snutch laboratories), I will present a preliminary analysis of 13 million human polyadenylated native RNA sequence reads for the GM12878 model cell-line. This analysis includes descriptions of full-length transcripts up to 22kb, alternative isoforms, polyA tail length, and RNA modifications. Additionally, ~24 million cDNA nanopore reads were obtained from the same RNA samples, which permit direct comparison of both transcriptome profiling methods.
Angela Brooks is an Assistant Professor of Biomolecular Engineering at UC Santa Cruz. Her research group focuses on identifying cancer genome alterations that disrupt gene regulation, particularly through RNA splicing. Angela has expertise in transcriptome sequencing analysis, cancer genomics, functional genomics, and bioinformatics. She received her Ph.D. from UC Berkeley and was a post-doctoral fellow at the Dana-Farber Cancer Institute and the Broad Institute.
Shaking up the short reads - using MinION sequencing to enhance an outbreak investigation
Antimicrobial resistance (AMR) is a global health concern and causes healthcare-associated infections with limited antibiotic treatment options. Outbreaks of healthcare associated pathogens can spread rapidly and silently particularly in the case of asymptomatic carriage. Methicillin-resistant Staphylococcus aureus (MRSA) has been a public health concern for decades and causes a multitude of infections including skin and soft tissue, bone and joint, pulmonary and blood stream. Hospitals in the United Kingdom have a ‘zero tolerance’ policy towards healthcare-associated MRSA blood stream infections, meaning the yearly ceiling for these cases is zero. Therefore, outbreaks can have major clinical implications for patients and financial penalties for hospitals. Rapid and high-resolution investigation using next-generation sequencing technology can inform outbreak investigations and aid clinicians in bringing them to a close. During February 2018 Infection Control clinicians in our hospital identified an increase in MRSA positive carriage swabs in patients who had been negative during admission screening. All positive cases were located on two adjacent wards. The wards did not mix patient or staff populations but shared an equipment area. We used Oxford Nanopore Technologies MinION sequencing device to obtain rapid long-read MRSA genomic data for nine positive screening swabs. We performed a proof of principle investigation to determine whether robust, informative data could be obtained in a clinically-relevant turnaround time that would determine the extent of the outbreak and the relatedness of the cases involved. Using the MinION we were able to report sequence data defining the outbreak within five working days of initial culture. The data enabled exclusion of two isolates based upon genomic differences and determined that the remaining isolates were likely to be acquired via recent transmission. We used two methods of data analysis which produced comparable results in line with epidemiological data. We were able to report the data faster than the reference laboratory and in a clinically relevant turnaround time. Use of rapid sequencing technologies is beneficial in outbreak situations and could save hospitals time and money. However automated analysis pipelines have not yet been developed which currently limits their accessibility and practicality in clinical settings.
Hayley Wilson is a post-doctoral research associate for Dr Estée Török in her newly established research group at the University of Cambridge. She completed her PhD in 2016 under the supervision of Professor Sharon Peacock, also at the University of Cambridge. She is currently working on investigating the carriage, transmission and infection of multidrug-resistant bacteria in various patient groups at the Cambridge University Hospitals NHS Foundation Trust. Her work utilises genomic techniques to provide high resolution data in investigating these pathogens and has recently begun working with long-read data produced by the Oxford Nanopore MinION.
Soup and nuts: nanopore applications
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.
The beauty and the beast
The Netherlands and tulips have for a long time been tightly linked and tulip agriculture is flourishing in our country. But tulip breeding isn’t without problems. Tulip breeding takes a long time and going from tulip seeds to a commercial product takes around 25 years. Pesticide use in tulip agriculture is relatively high so identifying traits that confer resistance to pests is an important issue. Having a tulip genome sequence will assist breeders in a more targeted breeding strategy. The genome of most tulip species is between 20 -35 Gbp which has prevented the sequence and assembly of this genome up until now. Sequencing and assembly of large genomes in general is a real challenge, but with the arrival of the alpha/beta PromethION, producing the sequence data for such a project has become easier. The next challenge is to order all this sequence data in a genome sequence. The long-read scaffolder/assembler Tulip was developed to do this in a reasonable amount of time. I will be discussing the sequencing and assembly of Tulipa gesneriana ‘Orange Sherpa’ using Tulipa-julia, the successor of Tulip.
Hans Jansen is CTO at Future Genomics Technologies based in Leiden in the Netherlands. A molecular biologist with broad experience, his focus has been on the use of next generation sequencing and bioinformatics for genome assembly and transcriptome analysis. As CTO he is responsible for the translation of academic knowledge and novel technologies into usable applications, providing early and easy access to these applications.
Leading the implementation of the MinION at ZF screens, he has been a member of the MARC consortium since it started in 2014. This group was formed to provide independent evaluation of the platform, pool data and analysis techniques, and exchange ideas for how to improve the sequencing protocol or bioinformatic processing.
Having used the MinION, GridION X5, and PromethION in their various genome projects, Hans has a wealth of experience in using the growing number of tools available for nanopore reads, and all from a biologists view point.