WYMM Tour: Perth

Effective cancer management critically depends on dynamic monitoring, yet the invasiveness of tissue biopsies and the high costs and resource demands of imaging techniques limit their frequent use. Liquid biopsies, particularly plasma cell-free DNA (cfDNA), present a less invasive option for continuous monitoring of tumour mass and genetic evolution. However, short-read sequencing technologies, which are predominantly used, have limitations in identifying DNA methylation, which can be explored to detect tumour-derived reads in sequencing data. Current short-read sequencing technologies do not directly identify DNA methylation patterns, and can only provide methylation information if combined with enzymatic or destructive chemical modification of DNA samples. Nanopore sequencing, in contrast, directly deciphers methylation patterns from the sequencing signal, enabling detailed mapping of cancer-specific methylomes, which can be leveraged to accurately quantify tumour fraction in plasma cfDNA samples. We are employing Nanopore sequencing to acquire high depth whole-genome methylomes from liver cancer specimens obtained from participants in a prospective Western Australian cohort study. Tumour-specific unmethylated loci for each participant are annotated and subsequently utilised to quantify tumour reads in longitudinal cfDNA samples using a deconvolution approach. This method can effectively estimate tumour burden from liquid biopsies. Correlation with contemporaneous radiological results will reveal its effectiveness as an alternative approach to track liver cancer evolution in a high frequency and non-invasive fashion.

We are a small group studying the impact of fungal diseases on plant physiology, at the Centre for Crop and Disease Management (CCDM). We apply high-resolution microscopy and spatially resolved multi-omic techniques to characterise the molecular basis of the phenotypes in diseased leaves. Our journey with Oxford Nanopore started with helping organise and also attend the hands-on Nanopore Workshop in Perth in June 2023. Although CCDM has many avid experienced users of Nanopore workflows, we are relatively new to the wet-lab protocols and downstream data analysis. I will present our troubleshooting of DNA extractions from devastating fungal pathogen, Pyrenophora tere-teres (causes net form net blotch in barley), library preparation and analysis of transcriptional isoforms in barley and towards long-read amplicon sequencing and its application in monitoring soil health and wine making.

In this study, we have focused on characterising transposable elements related to neurodegenerative diseases. Our earlier studies have shown the impact of SVAs, L1s, and Alu repeats on human health and their role in developing neurodegenerative diseases. However, these studies have been based on short-read sequencing. The present study gives an overview of the transposable elements and opportunities for characterising different types of SVAs using Oxford Nanopore technology.

Currently, more than 50 neurological and neuromuscular diseases are associated with repeat expansions. As the number and complexity of these disorders continues to grow, accurate detection and sizing of these expansions will be essential for neurogenetic diagnostics. Here we present the utility of long-read sequencing in clarifying the genetics of three separate expansion disorders. Cerebellar ataxia, neuropathy and vestibular areflexia syndrome is associated with biallelic, intronic pentanucleotide expansions in the RFC1 gene. The locus exhibits substantial genetic variability. To further investigate this heterogeneity, we used a combination of flanking PCR, repeat-primed PCR and targeted ONT sequencing. This uncovered a high proportion of complex alleles comprised of multiple repeat motifs and identified three novel motifs within our Australasian cohort. Spinocerebellar ataxia 36 is associated with large intronic GGCCTG hexanucleotide expansions in the NOP56 gene. WGS data was screened for known STR expansions using the analysis tool STRipy. This identified a probable expansion at the NOP56 locus in a family with autosomal dominant ataxia. Targeted ONT sequencing was required to accurately size the 7kb GGGCCT expansion. CCG expansions in several genes have been associated with Oculopharyngodistal myopathy (OPDM). Using a combination of linkage studies, short-read WGS and targeted ONT sequencing, we identified CCG expansions in the 5’UTR of ABCD3 within three large Australian OPDM families and five unrelated UK and French OPDM families. Using targeted ONT sequencing we were able to confirm the presence of unmethylated, mono-allelic CCG repeat expansions ranging from 118 to 694 repeats in all tested cases (n=19). These findings exemplify the ability of long-read sequencing to overcome limitations inherent to short-read technologies and current diagnostic techniques, as well as identifying and characterising novel STR expansion disorders. Future implementation of long-read sequencing into diagnostic spaces will become pertinent to provide patients with accurate and definitive diagnoses.

The advent of third-generation sequencing (TGS) represents a significant shift in the field of genetic sequencing, enabling long-read single-molecule sequencing to overcome limitations of preceding short-read NGS platforms. Several studies have assessed the utilisation of TGS platforms in HLA genotyping, though many of these studies have described the high error rate as a major limitation to achieving a robust and highly accurate HLA typing assay. In 2021, Oxford Nanopore Technologies (ONT) released the high-accuracy sequencing Kit 14 and the MinION flow cell model R10.4.1, which were reported to achieve sequencing accuracies up to 99%. The aim of this study was to validate this novel high-accuracy sequencing kit for HLA genotyping coupled with an in-house full-gene HLA PCR assay. Comparison with historical data obtained using legacy flow cell models such as R9.4, R10.3 and R10.4 was also done to assess progressive improvement in sequencing performance with each sequential release. The workflow was validated based on data throughput, sequence quality and accuracy, and HLA genotyping resolution. An initial validation was performed using an internal reference panel of 42 samples representing all known allele groups, followed by data obtained from 111 routine sequencing batch runs since the implementation, to assess assay performance and further improvements. Furthermore, challenges arising due to data storage, assessment of barcode contamination and the utilisation for different HLA genotyping applications are discussed. The findings of this study highlight advantages of ONT sequencing kit 14/R10.4.1 for HLA genotyping, implementation requirements and the challenges faced by the routine diagnostic HLA laboratory.

Ocean Genomes is a partnership between Minderoo Foundation and the University of Western Australia (UWA) to accelerate and scale the production of openly accessible reference genome assemblies for marine vertebrates. We are working with the Vertebrate Genome Project (VGP) to generate and release high-quality reference genome assemblies for marine vertebrates. The core VGP assembly workflow combines PacBio HiFi and HiC scaffolding to generate chromosomal-level phased diploid assemblies. Nevertheless, despite consistently impressive contiguity for our bony fishes assembled to date, assembly gaps remain in most chromosomes. The ultimate target is to generate gapless telomere-to-telomere (T2T) assemblies of every chromosome. We are exploring whether we can harness the greater read length of ONT to fill in regions that HiFi reads are currently unable to assemble, taking us closer to the goal of T2T reference genomes. Here, I will present preliminary results from gap-filling high-quality fish genomes with ONT data. I will discuss how many gaps can be closed, possible trade-offs between contiguity and quality, and which additional genome regions we are capturing.

Plant Pathologists are increasingly being called on to have a broader skill set than ever before. Here we will explore the opportunities for sequenced based diagnostic assays for accurate, timely and informative plant disease diagnostics to support Western Australia’s biosecurity and agricultural industries. A recent focus on High Throughput Sequencing (HTS) has brought what has been a powerful research tool for over a decade to the forefront of plant pathology diagnostics and biosecurity where it is increasingly being used to support diagnostic outcomes in the laboratory. HTS can be used for pest and pathogen detection, genome characterisation, genomic epidemiology, and new test development. Here we will highlight the ways in which we are incorporating it into our day-to-day workflows with a focus on rapid library preparation and amplicon-based assays that are increasingly more accessible with Oxford Nanopore sequencing instruments and chemistries. The move from research stream to daily diagnostic requires consideration of documentation for accreditation of new assays that may result from this research. There are currently no minimal standards or guidelines available for diagnosticians to use to ensure HTS generates accurate diagnoses, no formally endorsed guidance for biosecurity diagnostics in Australia at this time and a lack of national systems in place to promote sharing of diagnostic data between biosecurity agencies. In a rapidly changing environment, we will showcase new assays that span the fields of entomology, virology and bacteriology with a focus on biosecurity.

Acute respiratory infections are one of the commonly presented conditions treated with antimicrobials. Despite this, they are extremely challenging to treat since bacteria has the potential to readily become antimicrobial resistant (AMR). This is particularly important for chronic airway diseases where lifelong colonisation with common AMR pathogens is common. With a lack of new antimicrobials, alternative treatments including bacteriophage (phage) therapy are currently being explored, but detailed characterisation of both bacteria and bacteriophage are needed before proceeding to clinical use. Long-read sequencing via Oxford Nanopore could rapidly generate high quality, complete genome assemblies, identify antimicrobial resistance and virulence genes, and identify regions of genomic plasticity across the pangenome