WYMM Tour: Copenhagen

Abstract: The widespread availability of short-read next generation sequencing has transformed the speed and cost of molecular diagnostic testing. Nevertheless, there remain a significant proportion of patients that receive either an incomplete or no molecular genetic diagnosis. Using exemplar cases, this talk will summarize our progress moving from targeted to whole genome sequencing (WGS) assays on a recently installed PromethION (Oxford Nanopore Technologies). In a family that presented with congenital diarrhoea, trichorrhexis nodosa and cleft palate, standard-of-care targeted short read sequencing identified a heterozygous pathogenic mutation c.2808G>A p.(Trp936*) in the autosomal recessive disease gene TTC37. Subsequent long-read WGS to identify a second pathogenic allele revealed a single heterozygous candidate variant, c.2634+679A>G, that was determined to be in trans. In silico predictions supported the creation a novel splice donor site (SpliceAI score of 0.98). Globin-depleted whole transcriptome RNAseq, in combination with short-read sequencing, validated the predicted novel splice junction and confirmed a molecular diagnosis of trichohepatoenteric syndrome. In a further pair of unrelated families, we investigated the recurrent de novo duplication, c.136_138dup p.(Leu46dup), identified in GNAS exon 1. Analysis of bulk genomic DNA (which removes the need for PCR amplification) enabled characterisation of methylation status at this imprinted locus. By identifying single nucleotide variants that tag the parental haplotypes we could determine the parent-of-origin of these mutations.

Adoption of single molecule nanopore sequencing, by diagnostic laboratories, promises to aid the analysis and interpretation of incompletely resolved molecular genetic investigations.

Human Leukocyte Antigen (HLA) typing of large donor registries and biobanks as well as acute single patient/donor samples remains expensive, slow, and logistically challenging, despite recent developments in the field. We have tested and validated a cost-effective, accurate and highly scalable method for typing specific genes in the HLA region. This enables HLA typing from 1 to 96 individuals simultaneously, using a targeted PCR and Native Barcoding kit from Oxford Nanopore Technologies.
Methods:
A primer set for seven HLA genes (HLA-A, B, C, DRB1, DQA1, DQB1, and DPB1) was developed to work in a multiplex PCR reaction. The resulting amplicons provide a possible four-field resolution of the HLA Class I genes and G-group resolution of the HLA Class II genes. The entire process, from DNA to HLA typing result, takes a total of 5.5-10.5 hours depending on the number of samples processed simultaneously. Data analysis was conducted using NGSEngine®-Turbo from GenDx (The Netherlands), with analysis time ranging from 1 to 5 min per sample.
Results:
Samples from 96 Danish registered stem cell donors were typed using this method. One locus out of 1128 analysed loci was inaccurately called homozygous, leading to an accuracy of 99.91%.
Conclusion:
The rapid turnaround, low cost and high accuracy make this new method highly relevant for HLA typing of large biobanks and donor registries as well as for acute single samples. HLA typing can be obtained within one day, with a cost per sample of approximately 6 euros when 96 samples are sequenced simultaneously.
Keywords: HLA, long-read sequencing, Nanopore, high-throughput, cost-effective, native barcoding

X-Linked Dystonia-Parkinsonism (XDP) is a mendelian neurodegenerative disorder. Recently, a polymorphic transposable element (TE) insertion in the 32nd intron of the TAF1 gene has been identified as the genetic factor responsible for this disease. The XDP-TE is associated with TAF1 mis-regulation, but the mechanisms behind this phenomenon remain elusive. We hypothesize that repressive epigenetic marks on the XDP-TE are key players in this process. Thus, here we aim to dissect the molecular intricacies that keep the XDP-TE at bay and identify how it triggers aberrant TAF1 expression, ultimately leading to XDP. Leveraging advanced sequencing techniques and XDP patient-derived iPSCs and neural progenitor cells, we employed CUT&RUN and Oxford Nanopore Sequencing to identify epigenetic marks on the XDP-TE. To illuminate what factors establish these marks, and their effect on gene expression, we did CRISPR inhibition of various candidate genes coupled with RNA sequencing. We demonstrate that ZNF91 - a TE-binding KRAB-Zinc Finger Protein - establishes H3K9me3 and DNA methylation over the XDP-TE in a cell type specific manner in patient derived cells. Moreover, removal of DNA methylation, or both H3K9me3 and DNA methylation, severely aggravates the XDP molecular phenotype, causing a reduced TAF1 expression and increased intron retention.

Structural variants (SVs) affect more of the cancer genome than any other type of somatic alterations and include aneuploidies and rearrangements of the genome that can span up to megabases in size. SVs can manifest as simple events such as deletions, duplications and translocations, and more complex events such as chromothripsis or breakage fusion bridge cycles. Accumulation of SVs in cancer genomes is associated with aggressive disease, and the first targeted anti-cancer therapies were developed against the proteins affected by specific SVs. SVs have been challenging to resolve using standard short-read sequencing due to their often complex nature and when occurring in difficult-to-map regions of the genome. Here, we applied Oxford nanopore long-read sequencing (LRS) to resolve SVs in cancer genomes using both whole-genome sequencing and single-cell sequencing approaches. We demonstrate how nanopore-based genomic SV detection can be combined with methylation analysis to identify biallelic inactivation events involving genetic inactivation and epigenetic silencing. Using nanopore sequencing, we also detect complex SVs at single-cell resolution in brain tumours, and demonstrate how this can be leveraged to identify clonal selection during tumour progression.

Our research explores molecular signatures in breast cancer, including single-base substitutions, insertions/deletions, copy number variants, and structural variants. This pilot research study focuses on four high-risk familial breast cancer patients, previously analyzed using short-read sequencing from Illumina. By utilizing Oxford Nanopore’s PromethION platform, we aim to assess the consistency and accuracy of mutational signatures identified by Illumina in these patients using long-read sequencing. Specifically, we are investigating whether Oxford Nanopore can identify key molecular signatures, such as somatic variants and structural rearrangements, discovered in the previous Illumina dataset. In addition, we aim to examine if long-read sequencing provides comparable or enhanced utility in detecting complex genomic events. This comparison will determine if long-read sequencing is an important tool in cancer genomics research.

Several neurological disorders are caused by expansion of short tandem repeats (STRs). The homogeneity of long STRs makes it challenging to determine their structure with short read sequencing. Nanopore sequencing with adaptive sampling provides a rapid, cost-effective and flexible method for generating sufficiently long reads of all STRs of interest. We developed Abacus to quantify expanded STRs and visualize their complex structure. The combination of adaptive sampling and Abacus provides a rapid and customizable method with an easily interpretable output and thus a good platform for potential diagnostic use.

Several neurological disorders are caused by expansion of short tandem repeats (STRs). The homogeneity of long STRs makes it challenging to determine their structure with short read sequencing. Nanopore sequencing with adaptive sampling provides a rapid, cost-effective and flexible method for generating sufficiently long reads of all STRs of interest. We developed Abacus to quantify expanded STRs and visualize their complex structure. The combination of adaptive sampling and Abacus provides a rapid and customizable method with an easily interpretable output and thus a good platform for potential diagnostic use.

Identification of genomic rearrangement by microarrays or short-read sequencing often lacks information about the exact architecture and breakpoints of variants due to technical limitations. In addition, validation of complex structural variants (SVs) is often performed using custom assays, complicating confirmation of relevant findings. In this study we have used nanopore whole genome sequencing in patients with autism spectrum disorders to resolve complex chromosomal rearrangements that could not be resolved using short-read sequencing. For verification of complex SVs, we evaluate nanopore adaptive sequencing, targeting breakpoint. MinION sequencing of SV targets resulted in 13.5-17.8 Gb of data per flow cell, with mean on-target coverage of 18-34X and an off-target read depth average of 4.6X. Similar results were achieved using multiplexing of four samples on PromethION. We further show that background rejected reads can be used to detect SVs in non-targeted regions of the genome. Our results show that nanopore sequencing is an excellent technology both for resolving and validating structural variants, and that adaptive sequencing represents a robust, flexible and rapid strategy for verification of clinically relevant complex genomic rearrangements in patient samples. By providing sequence information, read depth and methylation data, nanopore adaptive sequencing has advantages over other validation assays for structural variants used in diagnostic laboratories today.