Highlights from NCM Houston
Future diagnostic potential for long-read sequencing as a single assay for imprinting disorders
Cate Paschal (Seattle Children's Hospital & University of Washington, USA) introduced her plenary talk by describing her research on Prader Willi syndrome (PWS) and Angelman syndrome (AS), which are complex, imprinted disorders associated with multiple anomalies. Describing the chromosomal region 15q11.2q13 as her ‘favourite region of the genome’, Cate explained it is also critical to these syndromes: the absence of maternally expressed genes in this region is linked to AS, while the absence of paternally-expressed genes is associated with PWS. Motivated by the fact it can take months to years, and multiple technologies, to detect the deletions, uniparental disomy, and imprinting defects commonly associated with these disorders, Cate and her colleagues investigated the potential use of nanopore sequencing to detect these variants in a single experiment.
‘There is huge potential for long read data with the integration of methylation calling’
Cate highlighted that it is not possible to determine if variants are on the maternal or paternal chromosome using short-read sequencing. Using PCR-free nanopore sequencing, native DNA of any length is directly sequenced, meaning base modifications can be detected alongside nucleotide sequence, and ‘large segments of the genome can be phased accurately’ — all from the same dataset.
‘We can show the differences in methylation on these different haplotypes, and having all of this data together in one assay is really powerful’
Using the high-output PromethION device to perform whole-genome sequencing of PWS and AS research samples, Cate shared how nanopore sequencing was able to simultaneously provide information on CNVs, SNVs, small deletions, methylation status, and parent-of-origin events from a single dataset, showing complete concordance with previous traditional analyses which require the use of multiple methods. Cate explained they intend to use nanopore sequencing to investigate other types of PWS and AS variants, and further expand the method to other imprinting disorders. Concluding, her talk, Cate described the potential future clinical utility of nanopore technology, expressing her view that ‘long read sequencing could streamline the testing process for imprinting disorders’.
Complex phased variants in inherited retinal diseases with long-read sequencing
Debarshi Mustafi (University of Washington, USA) described how understanding the molecular basis of inherited retinal disease (IRD) requires effective characterisation of mutations across over 300 genes. However, studies indicate that currently used short-read panel sequencing leaves 30–40% of patients with a non-diagnostic result. This is because short-read panels do not cover all relevant genes, miss intronic variants, and preclude the ability to perform haplotype phasing.
Debarshi and his team are investigating the potential of long nanopore sequencing reads, with real-time, bioinformatics-driven enrichment via adaptive sampling, to address these challenges. He described how their method allows the sequencing of IRD-related genes to sufficient depth of coverage ‘to do haplotyping, variant calling, methylation calling — everything — to identify disease variants’ on a single MinION Flow Cell, without the need for trio sequencing. In one example, clinical research samples from two subjects each featured three mutations in ABCA4. In the first, phasing of the long nanopore reads revealed the two severe mutations to be in trans, whilst in the other they were in cis, explaining the early onset sight loss versus late-onset, mild manifestations of the same disease, respectively.
‘Targeted long-read sequencing can provide comprehensive information of genetic variants contributing to disease’
Next, the team expanded their workflow to target ‘every gene that we can think of’ related to IRD, enabling the sequencing of 373 genes to ~20x depth on a MinION Flow Cell. Whilst the currently used clinical workflow requires multiple steps across months, Debarshi expressed his view that nanopore sequencing has the future potential to enable rapid, phased mutation analysis in a single step, from sequencing of the subject alone — in a matter of days or hours. He shared an example in which 11 hours of nanopore sequencing was sufficient to phase mutations in a clinical research sample. In another research sample, where only one variant had been identified via a validated clinical assay, nanopore sequencing revealed a second, missed pathogenic variant.
Debarshi is also investigating the potential of this technique to rapidly identify pathogenic variants in retinoblastoma, for which rapid diagnosis is critical. Sequencing research samples with adaptive sampling, they demonstrated the potential to characterise and phase mutations in the target RB1 gene — and also assess methylation. Concluding his talk, Debarshi emphasised the potential future utility of this method, and how the use of MinION ‘makes this accessible to any lab’ and ‘presents an evolution in addressing the missing heritability in retinal diseases and will be applicable to many other Mendelian disorders’.
Improving bacterial disease public health testing with nanopore sequencing
Kimberlee Musser (Wadsworth Center, New York State Department of Health, USA) began her talk by introducing the public health laboratories across New York State and their role in public health testing to determine the source of disease outbreaks, as well as national surveillance of diseases, such as influenza and Mycobacterium tuberculosis. She went on to explain how nanopore sequencing has been implemented at the Wadsworth Centre to detect antimicrobial resistant (AR) genes and analyse plasmids, as well as bacterial species identification, and rapid drug resistance detection for TB cases.
Kimberlee highlighted that nanopore sequencing has a huge benefit for characterising mobile genetic elements carrying AR genes, such as plasmids, and the genomic context to investigate plasmid transfer events. Due to the long nanopore reads, plasmids carrying AR genes can be fully characterised and the AR genomic context identified to inform outbreak investigations. Kimberlee explained that due to the repetitive regions in bacterial genomes, long reads are required to sequence complex regions, to identify the location of the AR gene and determine whether it is in the bacterial genome or the plasmids — which cannot be achieved with short-read sequencing.
Kimberlee outlined multiple examples of how nanopore sequencing has been implemented in the laboratory to characterise AR genes in disease outbreaks across the USA, including characterising a novel AR gene to determine genomic context for future reference. Kimberlee also detailed how nanopore sequencing was used to reassess previous samples of Vancomycin-resistant Staphylococcus aureus (VRSA) to identify the donor gene that created the VRSA strain.
Kimberlee also highlighted how she and her team have been developing an M. tuberculosis complex (MTBC) test to analyse drug resistance in TB research samples using nanopore sequencing. With nanopore sequencing, they can detect drug-resistant MTBC up to 15 days earlier and in some cases, they can have initial results available in less than 24 hours. This workflow also sequences more targets than their previous assay.
Kimberlee ended her talk by outlining her plans for nanopore sequencing in public health research, including for non-tuberculosis mycobacteria identification and direct detection of Legionella and Shiga-toxin producing Escherichia coli in water, stool, respiratory, and autopsy samples.