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.

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.

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.

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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

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.

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.

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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.