WYMM Tour: Zurich

Reconstruction of complex genomic regions containing recent segmental duplications, repeat expansions or complex structural variants has been challenging using short read sequencing methods. These regions, however, potentially harbor a substantial fraction of disease-causing variants missed in unsolved rare disease cases (still around 50% of all cases). The introduction of Nanopore long-read genome sequencing (LR-GS) into clinical genetic testing promises to reduce this gap by resolving complex genomic regions, facilitating haplotype-phasing and revealing complex SVs that often consist of closely located inversions, tandem-duplications and deletions. Therefore, a study group consisting of four German university hospitals was launched aiming to evaluate the added diagnostic value of Nanopore LR-GS and to demonstrate its sustainability in clinical practice. The study group relies on established collaborative structures with multidisciplinary teams, data analysis task forces (DATFs), and data interpretation task forces (DITFs). We analyzed a cohort of solved cases featuring causal repeat expansions, variants in duplicate genes, complex rearrangements, and aberrant DNA methylation. We included multiple GiaB samples to benchmark variant detection methods for accreditation of LR-GS. The experience gain from the initial pilot serves as a blueprint for the analysis of a larger clinical cohort (>1000 samples). In this talk I will focus on our experience with the bioinformatics analysis of Nanopore LR-GS data for rare disease diagnostics, including development and/or application of tools and pipelines for SNV, indel and SV detection, haplotype phasing, length estimation for repeat expansions, genotyping in duplicate genes and haplotype-specific DNA methylation analysis. I will discuss our quality criteria and benchmarking efforts and demonstrate the need for generating large Nanopore LR-GS background dataset to facilitate systemic filtering and efficient clinical interpretation of SVs in clinical diagnostics.

The World Health Organization has declared antibiotic resistance one of the ten most severe global health threats, with resistant infections leading to higher mortality and morbidity. Real-time genomic technology has the potential to speed up pathogen and antibiotic resistance profiling in the clinical setting. This is especially relevant for infections with carbapenemase-producing Enterobacterales (CRE), which present a significant resistance burden due to their ability to hydrolyze a variety of antibiotics. Here, we directly compared pathogen and resistance predictions between current clinically established and real-time genomic approaches, using all CRE infection cases at the Technical University of Munich Rechts der Isar hospital in 2023 as an example. We have further leveraged this unique dataset for rapid predictions of plasmid-encoded antibiotic resistance, which is essential to monitor antibiotic resistance spread via horizontal gene transfer. Our preliminary results confirm high concurrence between both approaches while highlighting the advantages of real-time genomics in terms of additional relevant infection control information through plasmid detection and annotation.

Background/Objectives: Short-read sequencing (SRS) is the most widely used technology in clinical sequencing, but it has limitations in areas of the genome harbouring complex structural variants (SVs) or repetitive/homologous sequence, and variant phasing information is often lost. Long-read sequencing (LRS) technologies should not suffer these challenges and have improved to the point of feasible use in clinical sequencing. Thus, we assessed the latest LRS technologies for their ability to detect challenging SVs. Methods: Four DNA samples with challenging SVs as well as single nucleotide variants previously detected by SRS (PCR-free WGS, PE150, 60×) were sequenced on a PacBio Revio as well as on an ONT PromethION platforms (30×) and analysed with their respective standard pipelines. Several further SV callers were applied and regions of interest were manually reviewed using Integrative Genomics Viewer. Results: Our data revealed differences in the performance of the tested LRS platforms and SV callers for challenging variants, including complex SVs and SVs with breakpoints in non-unique sequence regions. Nevertheless, both LRS approaches were able to characterise breakpoints expected by SRS and to phase sequence variants according to their respective read lengths. Conclusions: LRS can be the solution for breakpoint identification in repetitive regions and for characterisation of complex SVs not exceeding their read length. Both improved SV callers and improved read length are vital to take full advantage of LRS in clinical genetic testing. Current LRS technologies achieve SRS-like error rates at affordable prices and so enable an improved future for clinical sequencing.

Direct RNA sequencing offers the possibility to simultaneously identify canonical bases and epi-transcriptomic modifications in each single RNA molecule. Thus far, the development of computational methods has been hampered by the lack of biologically realistic training data that carries modification labels at molecular resolution. We have recently reported the synthesis of such samples and introduced mAFiA (m6A Finding Algorithm) that accurately detects single m6A nucleotides in both synthetic RNAs and natural mRNA on single read level. Following up on this, we have extended mAFiA to all 18 DRACH motifs and detection of pseudouridine (psI-co-mAFiA). psI-co-mAFiA identifies shared and disease-specific differential modification site in mouse models of cardiovascular diseases on single-site and single-read level.

The demand for long-read sequencing has been steadily increasing due to its wide range of potential applications. Oxford Nanopore Technologies (ONT) has emerged as an accessible tool for this purpose. However, this advancement comes with certain challenges, particularly in ensuring the quality and quantity of analytes, as well as in bioinformatics analysis. At Microsynth we have extensive experience utilizing ONT and have encountered and addressed these challenges. In this presentation, we will share successful applications of ONT for the analysis of plasmids, bacterial and eukaryotic genomes, microbiome, viruses, and genetically modified cells. Additionally, you get insights and strategies for overcoming some challenges in long-read sequencing analysis.

Genetic diagnostics of repeat expansion and contraction disorders, including hereditary ataxias and facioscapulohumeral muscular dystrophy (FSHD), respectively, present significant challenges due to their phenotypic overlap and genetic complexity. Traditional methods lack precision and fail to capture crucial epigenetic markers. To overcome these limitations, we implemented nanopore Cas9-targeted sequencing for repeat expansions, enabling precise analysis of 10 repeat loci associated with hereditary ataxias. This method allowed for parallel repeat length, sequence, and methylation detection. Application of this approach to 100 undiagnosed ataxia patients revealed causative repeat expansions in 28 individuals, with RFC1 biallelic expansions emerging as the most common etiology. Moreover, identifying a novel repeat motif underscores the diagnostic potential of nanopore sequencing in uncovering previously unrecognized genetic variants. In the context of repeat contractions in FSHD, conventional diagnostics often fail to capture the full spectrum of genetic and epigenetic factors associated with this disease. To that end, we employed Cas9-targeted nanopore sequencing to characterize permissive haplotypes, repeat size, methylation profiles, and structural variants in FSHD patients. As proof of concept, we clarified the unexpectedly high average distal methylation in a paucisymptomatic patient homozygous for a permissive haplotype, harboring a repeat contraction with two repeat units on one allele. The ONT data revealed the presence of the contraction as a mosaic, causing an increase in the average percent methylation. In another patient, resolving a complex hybrid allele revealed the presence of a hypermethylated rare 4qC166H haplotype, which makes FSHD unlikely as the reason for the patient’s symptoms. Overall, our work showcases the power of nanopore sequencing in providing comprehensive insights into the genetic and epigenetic landscape of repeat expansion and contraction disorders and in uncovering novel disease mechanisms.