Nanopore Day, Utrecht
Wed 27th November 2019
UMC Utrecht, Utrecht, The Netherlands
Hear about the latest tech updates for Oxford Nanopore as well as talks from local scientists about their latest work using nanopore technology.
Confirmed speakers include:
- Ies Nijman, Manager Useq, UMC Utrecht
- Federica Brogioli, Orvion
- Sander van Boheemen, Erasmus MC
- Reindert Nijland, Assistant Professor Molecular Marine Ecology, Wageningen University
- Anna Dolnik, Charité Universitätsmedizin Berlin
- Christina Stangl, UMC Utrecht
- Leila Luheshi, Associate Director, Clinical and Translational Research, Oxford Nanopore Technologies Ltd.
- Olivier Lucas, Associate Director, Regional Sales (Europe South), Oxford Nanopore Technologies Ltd.
There is no delegate fee for this event.
Your place at this event will be confirmed via email from firstname.lastname@example.org. Completion of this form does not constitute confirmation. Spaces are limited and will be allocated on a first-come, first-served basis.
The agenda is yet to be confirmed.
Aim: Viral genotyping is an important tool to guide and monitor treatment decisions, support and evaluate (hospital) infection control measures, and contribute to public health surveillance of a broad range of diseases including viral hepatitis, enteroviruses and HIV. For example, the severity of hepatitis can vary between hepatitis B genotypes, and different patterns of resistance-associated mutations are involved in antiviral resistance for various genotypes for hepatitis C virus. Currently, routine genotyping is mostly performed using targeted amplification and subsequent Sanger sequencing, with target choice and performance characteristics tuned to specific use cases. The fast development of deep sequencing platforms offers potential for merging clinical and public health applications into a single combined application, thus potentially cost-saving when used in clinical laboratories with sufficient throughput for pooling of analyses.
Method: We have developed a rapid multiplexing and library preparation protocol using real-time nanopore sequencing to determine the genotypes of multiple viral targets. Thus far, these viral targets include HBV, HCV and measles virus. One nanopore run allows for simultaneous sequencing of multiple viral targets.
Results: Viruses from various species, genotypes and viral loads were rapidly sequenced on the Nanopore platform, demultiplexed and subtyped. All samples were confirmed by Sanger sequencing as gold standard. The reported high error rate associated with the nanopore sequencing chemistry was resolved by adequate read coverage.
Conclusions: We were able to accurately subtype a variety of viral species. Furthermore, multiple samples from different virus species were successfully combined, sequenced, and demultiplexed after one sequencing run. This allowed us to rapidly genotype viruses. Future efforts will determine the feasibility of nanopore sequencing in antiviral resistance testing.
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.
Acute myeloid leukemia (AML) is characterized by abnormal clonal expansion of malignant cells. A classification of patients is based on cytogenetics and molecular markers as described in the 2017 European LeukemiaNet (ELN) recommendations. Conventional karyotyping is still needed to determine many “high-risk” AML cases characterized by myelodysplasia related changes (AML-MRC) and this information is only available after 5 to 7 days at the earliest. Using Oxford Nanopore technology (ONT) we established rapid karyotyping based on low-coverage whole genome sequencing with library prep and bioinformatics analysis being performed within a timeframe of only 48 hours. An effective throughput, 10-15 Gb per flowcell, is sufficient to achieve 3-fold whole genome coverage (range 2.5-3.6) in order to reproduce results of conventional karyotyping with a resolution of 1 Mb. In parallel, DNA analysis allows to capture DNA methylation information that is characteristic for AML-subtypes and can be used for classification. To enable the discovery of structural variations, we established a complementary transcriptome sequencing protocol to allow the analysis of fusion genes in a similar timeframe. Using direct cDNA sequencing we could achieve up to 5 million reads on a single flowcell in 20 hours which is sufficient to cover a balanced translocation t(9;22) resulting in expression of fusion gene BCR-ABL1 in the model cell line K-562 with 50-80 reads resulting in robust detection of the fusion. Combined ONT based CNV profiling, methylation workflow and fusion gene detection provides the opportunity to rapidly characterize AML at the genomic level, which is needed for improved genotype-based treatment strategies.
Two examples of environmental applications of MinION technology. An industrial wastewater treatment plant has sporadic issues with bulking sludge. Using MinION sequencing we monitored the microbial biodiversity of this WWTP during approx. 2 years. We have identified a number of bacteria that might be causing poor sludge settling or might be beneficial. These will be monitored more closely in the coming year to try and optimise the WWTP and improve sludge stability. An industrial site is contaminated by pollutants such as chlorinated compounds and fuel oxygenates. Using MinION sequencing we investigated the potential for biological natural attenuation. We have confirmed the presence of bacterial species known to be involved in the degradation of such compounds. The specific bacteria are more abundantly present in the sites that are contaminated compared to the clean references, meaning that they are growing using those compounds. These results will be used to start a bioremediation process and select interesting qPCR targets for monitoring.
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