WYMM Tour: Brisbane

Lung cancer is a common cancer with multiple subtypes. To date, the majority of genome studies of lung cancer have been conducted using short-read sequencing. Long-read sequencing using the Oxford Nanopore Technologies enables direct sequencing of DNA allowing simultaneous whole genome sequencing and methylation profiling. We have used the promethION to perform whole genome sequencing of samples that represent different tumour purities to understand the performance of the technology on clinical tumour samples. We then used whole genome sequencing of a cohort of lung cancer biopsy samples to characterize the somatic mutations and determine whether long read sequencing offers additional insights compared to short read sequencing. This talk will summarize our recent findings and experiences with long read sequencing.

DNA replication stress is a hallmark of cancer that is exploited by chemotherapies. Current assays for replication stress have low throughput and poor resolution whilst being unable to map the movement of replication forks genome-wide. We present a new method that uses nanopore sequencing and artificial intelligence to map replication forks and measure their rates of movement and stalling in melanoma and colon cancer cells treated with chemotherapies. Our method can differentiate between fork slowing and fork stalling in cells treated with hydroxyurea, as well as inhibitors of ATR, WEE1, and PARP1. These different therapies yield different characteristic signatures of replication stress. We assess the role of the intra-S-phase checkpoint on fork slowing and stalling and show that replication stress dynamically changes over S-phase. This method requires sequencing on only a single nanopore flow cell, and the cost-effectiveness and high throughput enables functional screens to determine how human cancers respond to replication-targeted therapies.

As an integral aspect of their digestive process, ruminants, including cattle, generate greenhouse gases (GHGs), notably methane. In 2022, livestock farming contributed to a substantial 11.1–19.6% of total GHG emissions, solidifying its position as a significant anthropogenic source of GHGs. Enteric methane, a prevalent by-product of livestock farming, significantly contributes to global warming. The escalating global population, coupled with an increasing demand for protein-based diets, anticipates the continued expansion of livestock ranching, intensifying concerns about the adverse environmental impact caused by methane emissions. To counteract these detrimental effects, concerted research efforts have been directed towards fostering more sustainable farming practices, dietary adjustments for livestock, and the exploration of innovative technologies designed to capture and redirect methane emissions from livestock farming.

Within the digestive system of ruminants, a consortium of microorganisms thrives in the first compartment of their specialized stomach, the rumen. These microorganisms facilitate the breakdown of complex carbohydrates through the enteric fermentation process. Methanogenic microbes harness the hydrogen produced during fermentation to reduce carbon dioxide, eventually yielding methane as a by-product. The process of rumination, where ruminants regurgitate cud from the rumen to the mouth, indicates a potential association between the oral and rumen microbiomes and the methane emission phenotype. Several studies have successfully predicted methane emissions from ruminant oral microbiomes. Recognizing the pivotal role of the oral and rumen microbiome’s composition and activity on methane yield, our study hypothesized the potential mitigation of ruminal methane emissions by strategically selecting animals with a microbiome demonstrating a reduced propensity to produce methane. We conducted a comparative analysis of microbiome signatures and activity profiles between oral and rumen samples using Oxford Nanopore sequencing technologies. The longer reads generated by ONT sequencing retained the continuity of read information, benefiting the in-depth annotations for both taxonomical and functional profiles of the metagenome. Our results demonstrated an innovative approach to infer low methane-emitting cattle via a non-invasive oral microbiome and an integration into existing breeding programs, maximizing the benefits of selective breeding.

Low- and Middle-income countries (L&MICs) are disproportionately impacted by epidemics and consequently suffer from severe, potentially long-term effects to public health and economic outcomes. This was made abundantly clear during the recent COVID-19 pandemic where such countries faced unique challenges during the course of the pandemic but also continue to suffer from long lasting poverty and health crisis in direct response to it. There is a clear and urgent need to better develop health and medical services within L&MICs to alleviate some of the pressures they encounter during and in response to disease outbreaks. Genomic epidemiology achieved through expansive sequencing efforts, offers an opportunity to redress some of these distinct challenges. Currently such sequencing is difficult to deploy at the scale necessary in L&MICs for a host of reasons, not least among which is the difficulty in establishing effective sequencing facilities forward into otherwise unsupported outbreak areas. Existing logistics, infrastructure, resources and trained personnel are often lacking so the ability to miniaturise, pre-train and pre-deploy independent sequencing services is essential. The ARTIC Network was established in 2016 in response to the Ebola Virus outbreaks of West and Central Africa where a clear need to expand real-time “frontline genomics” was identified. In service of this ARTIC develops, curates and deploys a suite of tools to more than 40 partnered institutes across 20 L&MICs, with all protocols and digital tools provided openly online. Oxford Nanopore Technologies has been adopted as a core element of this initiative, having been used to design robust sequencing protocols, analysis tools and portable laboratories that can be employed in otherwise arduous scientific environments. These tools were extensively utilised during the recent pandemic and represented a good first step in closing the disparity faced by L&MICs.

The lifecycle of an mRNA vaccine begins with its manufacture and formulation into lipid nanoparticles. The mRNA is then delivered to patients, where it is taken up by recipient cells and translated into the encoded protein in the cytoplasm. This study uses Oxford Nanopore (ONT) cDNA and direct RNA sequencing to analyze each step in the mRNA vaccine lifecycle. We first show that ONT sequencing can be used during mRNA manufacturing to monitor mRNA quality and integrity, and to detect contaminating RNA species that can induce unwanted inflammatory responses. Direct RNA sequencing is also used to detect modified nucleotides and analyze mRNA chemistry. In addition, long-read sequencing can directly measure the impact of different degradation pathways on mRNA integrity during formulation and storage. Finally, we describe the uptake, expression, and final degradation of mRNA vaccines within cells. We sequence the mRNA vaccine within the recipient cell and investigate its broader impact on gene and protein expression. Together, this study uses ONT sequencing to trace the lifecycle of an mRNA vaccine, providing quality data from throughout the manufacturing process and insight into its mode of action within the cell.

The structure of RNA has been shown to be critical to its function, whereby specific structural motifs can affect stability, splicing, translation and RNA-protein binding interactions for coding and non-coding RNA species. Additionally, highly organised structural domains enable RNA species to function beyond the control of gene expression, including scaffolding of complex assemblies and molecular chaperoning. Importantly, RNA structure is dynamic and growing evidence implicates RNAs as active sensors of their local environment, undergoing conformational changes in response to environmental stimuli. In neurons, precise spatiotemporal expression of RNA has been shown to be critical for key neuronal processes including cell development/identity, cell signalling and synaptic plasticity. The role of structural motifs and domains in key RNAs is predicted to have a significant influence on their function/s in these important neuronal processes. Here I have begun to interrogate the structural dynamics of the neuronal RNA population. Utilising chemical SHAPE probes and direct RNA sequencing, I have identified activity dependent structural states in neuronal RNAs that may inform novel mechanisms for posttranscriptional gene regulation.

Schizophrenia is a complex polygenic disorder with several hundred genetic variants contributing to disease risk, most of which are located in non-coding regions of the genome. These genetic variants affect the regulation of gene networks and biological pathways underpinning core developmental cellular processes beyond just synapses and neurotransmitters. Our multi-omics analysis, including Oxford Nanopore Technology sequencing of schizophrenia-derived olfactory stem cells, reveals distinct epigenetic profiles in patients. These epigenome signatures help explain some transcriptional and cellular dysregulations associated with the disease. Our goal is to develop a systems-based approach to identify new target molecules for novel RNA-based gene therapies for schizophrenia.

Myeloproliferative neoplasm (MPN) is a chronic blood cancer characterised by the overproduction of mature blood cells like erythroids, platelets or megakaryocytes. To date, there is no cure for MPN patients, and current treatments reduce the patient’s risk of thrombosis or stroke. Additionally, patients have to live with the emotional burden of being at risk of disease progression to a treatment-resistant and rapidly lethal acute myeloid leukaemia (AML), with an overall survival of less than one year. MPN is the consequence of acquired mutations in blood-forming stem cells. While we understand that specific mutations and increasing genetic heterogeneity determine a patient's risk of disease progression, we don’t know why. Understanding the consequences of genetic heterogeneity at single cell resolution and its role in disease will offer valuable insights and potential therapeutic directions.

Recent technological advancements, such as single cell gene expression and DNA genotyping, have facilitated the deconvolution of tumour heterogeneity. However, there has been a gap in linking these techniques to infer the effect of mutations on gene expression. To overcome this, we have developed a novel single cell pipeline, combining targeted gene capture with Oxford Nanopore Technology (ONT) long-read sequencing to genotype 30 key myeloid cancer-associated gene transcripts from 10X Genomics single cell RNA sequencing data.

This helps us understand how disease-causing cells behave differently from healthy cells, with the hope of finding a way to eliminate these harmful cells. This knowledge will be invaluable in optimising the use of both existing and novel cancer therapies, ultimately leading to improved patient outcomes.

The centre for tropical bioinformatics and molecular biology has been running small grants to bootstrap nanopore projects in tropical North Queensland since 2019. Each year we have seen a growth in the diversity and level of innovation in grant ideas from applicants, and while not everything has been successful we have learned a great deal about what works and what doesn’t. In this talk I will showcase innovative applications of nanopore sequencing developed by our researchers. These applications will highlight how different capabilities of nanopore sequencing (long reads, fast turnaround times, methylation detection) have been particularly valuable for applications to tropical biodiversity and when rapid decisions are needed in the field.

As most marine microbes have not been cultured, the establishment of large databases of metagenome-assembled genomes (MAGs) have markedly improved our understanding of marine microbiomes. However, such databases seldom include Australian oceans or coral reefs globally. These databases have also mainly focussed on marine prokaryotes, though MAGs from ubiquitous and dominant lineages like Pelagibacter, Prochlorococcus, SAR86, etc are mostly absent, a phenomenon presumed to result from an inability of short reads to resolve these strain-diverse populations. To overcome these difficulties, the Great Barrier Reef Microbial Genomes Database (GBR-MGD) was established by subjecting seawater from across the Great Barrier Reef in Australia to Nanopore/Illumina hybrid sequencing, hypothesizing that long reads could span strain-variable regions and enhance MAG recovery. These efforts generated >5,000 prokaryote MAGs (~1,500 near-complete), including complete Pelagibacter, Prochlorococcus, and SAR86, showing that even “difficult” taxa are reliably recovered, and often circularised, in Nanopore hybrid metagenomes but not those using Illumina short reads only. We show that this platform-specific phenomenon results from not only an inability of illumina’s short reads to resolve strains, but multiple systematic biases inherent to the Illumina platform. Our hybrid strategy also facilitated the recovery of several chromosome-level, telomere-to-telomere picoeukaryote MAGs and >100,000 viruses, including novel clades of marine Crassvirales. Leveraging the GBR-MGD, we find that specific microbial taxa can reliably predict the effects of reef management practices using machine learning, such as the creation of refuge reefs that are closed to fishing. Hence, the GBR-MGD represents an unprecedented and holistic resource for marine researchers.