WYMM Tour: Bonn

Genetic diseases can be caused by different types of mutations. Most of these can now be identified by routine methods, e.g. (MS)-MLPA, microarray, exome sequencing and short-read genome sequencing. However, several methods are sometimes necessary to detect the different types of mutations. There are also limitations, e.g. in the exact determination of repeat lengths (RE), complex structural variants (SV) or paralogous genes. Using long-read sequencing technologies, it is now possible to overcome these limitations and detect all previously known mutation types with high sensitivity and precession. Thanks to improvements in throughput, costs and library preparation, long-read sequencing technologies can now be used routinely. Using various examples from routine diagnostics, the possible applications, advantages and disadvantages of nanopore sequencing are presented.

VNTR: Mutations in the coding sequence within large variable number tandem repeats (VNTR) can lead to different monogenetic diseases. These mutations are impossible to detect by short-read sequencing due to their repetitive nature, and long-read variant callers might fail to detect the actual sequence change due to mapping inconsistency and the variability of the motif composition. We developed a pipeline for the detection of mutations and motif compositions within VNTRs using Oxford Nanopore sequencing. Samples are sequenced, mapped and phased across the whole VNTR. The extracted reads from each haplotype are then corrected, trimmed, and a consensus sequence assembled de novo. Using the consensus sequence, the motif composition of the VNTR is constructed visually. Macrosatellite - FSHD: FSHD is a very common form of muscular dystrophy caused by changes within a highly complex and large macrorepeat (D4Z4-repeat at the DUX4-gene) at the telomeric region of chromosome 4. This presents many challenges for genetic analysis. We have developed a potential approach using Nanopore-sequencing to help navigate this region. This allows for a subsequent more in-depth analysis based

This talk highlights the results of sequencing 1,019 samples from the 1000 Genomes Project using ONT. Our analysis approach integrated linear and graph-based approaches for structural variant (SV) analysis using pan-genome graph augmentation. In total, we discovered 167,291 sequence-resolved SVs from different SV classes in the 1,019 samples, including deletions, duplications, insertions, and inversions. Our approach enabled a detailed characterization of mobile element insertions and complex inversions at single-nucleotide resolution and we could investigate potential SV formation and recurrence mechanisms that involve repeat sequences. Our open access dataset highlights the benefits of ONT long-read sequencing in characterizing polymorphic SVs at the population level.

Nanopore sequencing has multitudinous applications in the clinical context: Here, we shall focus on the use of nanopore sequencing during an outbreak of mpox and budding applications in the context of mRNA therapeutics and mRNA modifications. The first use case is tied to the more “classical” application of long read sequencing to resolve viral genomes and to span repetitive stretches are non accessible with short read sequencing. During the 2022 outbreak of monkeypox virus, we performed genomic sequencing of a local mpox sample on a PromethION and in parallel on an Illumina device. Both applications could recover most of the genome and SNPs. Only Nanopore sequencing could resolve two major structural variants in the genes OPG208 and OPG015. Reannotation supports both variants on the transcript level. In conclusion, nanopore sequencing could be used as a standalone sequencing technique and the technique could recover structure of monkeypox transcripts, that would otherwise have been missed. The second use case focuses on Direct RNA sequencing (dRNA-seq), to sequence native RNA. Such sequencing is helpful in the broader context of human diseases and mRNA therapeutics. Perhaps the most prominent example for the use of modifications in mRNA therapeutics is the incorporation of methylpseudouridine in mRNA vaccines to decrease immune response and increase yield. Additionally, there are budding applications in the context of translation-inducing readthrough drugs (TRIDs). This type of drug could be helpful in 10-20 % of clinical diseases, where a premature stop codon occurs. One possibility to facilitate read through is to apply pseudouridine directly to a stop codon. Recently, Schartel et al., have designed an organelle based targeting system for single pseudouridines. RNA004 dRNA-Seq was used in this context to evaluate site-specific pseudouridination. In the future this may also be used to verify disease-causing aberrations of methylation levels.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and the fastest-growing neurological disorder, with over ten million affected persons worldwide. Thus far, no cure and only treatments to manage the symptoms are available. Genetic and environmental factors can cause PD. Out of all PD cases, approximately 15% are caused by a pathogenic variant in a single gene (i.e., monogenic PD) or a strong risk factor. Although PD pathogenesis has not yet been fully elucidated, mitochondrial dysfunction and impaired mitochondrial quality control are considered key players. We demonstrated the applicability of Nanopore long-read sequencing to detect mitochondrial DNA (mtDNA) variations. We have established and validated Nanopore sequencing workflows to assess mitochondrial DNA methylation and somatic variants of the mitochondrial genome (i.e., heteroplasmy). In the context of PD, we investigated mitochondrial genome heteroplasmy in samples derived from individuals with PRKN- and PINK1-related PD. We found that mitochondrial heteroplasmic variant load is associated with disease susceptibility, where a higher number of heteroplasmic variants is associated with a higher risk of PD (Z=2.93, p=0.0033). Currently, we are expanding our research to other forms of PD to assess the implications of mtDNA variants in PD and explore potential gene-environment interactions.