WYMM Tour: Vancouver

I will discuss clinical and research uses of Oxford Nanopore long-read sequencing, including the development of control databases, and provide examples of clinically relevant variants missed by prior testing.

A key limitation of current genetic testing technologies is their inability to accurately predict parental segregation of germline variants without external data. We demonstrate that use of Parent-of-Origin-Aware genomic analysis enables precise parent-of-origin predictions for germline variants and has the potential to improve the care of patients and at-risk family members for hereditary cancer.

The Personalized Oncogenomics (POG) program is a precision medicine initiative integrating short-read whole genome and transcriptome analysis (WGTA) into the clinical care of advanced cancer patients. As part of the TFRI Marathon of Hope Cancer Centres Network, we have re-sequenced over 400 tumour samples from POG on the Nanopore platform. This long-read data, layered on the existing short-read WGTA, enriches our understanding of these advanced and metastatic tumours. Long-read sequencing resolves complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long reads make possible very long-range phasing, facilitating the exploration of multiple hits to tumour suppressors, discovery of allelically differentially methylated regions (aDMRs) and resolution of allele-specific expression. Promoter methylation in tumour suppressor genes is associated with clinical phenotypes including microsatellite instability and homologous recombination deficiency. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology. The value of long-read sequencing has led to the integration of this technology prospectively, where new POG samples with sufficient material are simultaneously profiled with short and long reads.

NTRODUCTION: Transplantation is a life-saving therapy but carries significant risks, including rejection, graft-versus-host disease, infection, and malignancy, which can lead to failure and death. These complications arise from the interaction between the recipient's immune system and the transplanted organ, requiring long-term toxic drugs to prevent immune injury. Traditional pre-transplant assessment focuses on 11 HLA genes, but advancing technologies now allow for the assessment of many more genes relevant to graft tolerance. We are using adaptive sequencing to preemptively identify patients at risk for lethal complications and to personalize treatment strategies.

RESULTS: We have established flexible adaptive sequencing pipelines that include traditional HLA genes, ABO, other blood group histocompatibility genes, pharmacogenomics loci, or whole chromosomal arms (e.g., 6p or Xp for loss of heterozygosity determination). By testing different BED files, run parameters, and flow cells (GridION, PromethION), we currently achieve up to 5.5x enrichment, which is sufficient for variant calling and LOH analyses. Our goal is to develop individual immune-genomic risk profiles for transplant patients and identify key polygenic predictors of treatment response to minimize toxic immunosuppression.

CONCLUSIONS: We have achieved dramatic success in reducing early immune injury through the combination of molecular diagnostics and structural biology. This research represents the next critical step to refine precision medicine, improve long-term patient outcomes, reduce healthcare costs, and enhance the quality and duration of life for transplant recipients in Canada and globally. Incorporating comprehensive genomic information into clinical decision-making will be crucial for reducing and eventually eliminating toxic immunosuppressive drugs through operational tolerance.

Infectious diseases comprise three of the top 10 leading causes of death globally. Typically, infections caused by bacteria can be treated with antibiotics; however, the overuse and misuse of antibiotics has caused dramatic increases in antimicrobial resistant (AMR) organisms. Bacterial AMR was directly responsible for 1.27 million global deaths in 2019 alone. With the increase in AMR and limits in new antibiotics being developed, we risk going back to the pre-antibiotic era where common medical procedures can be deadly. To tackle this problem, the Lee Lab, as a part of the AVENGER (Advanced LNP RNA Vaccines Engineered with Next-Generation designs to Enhance pandemic Readiness) team, will be using ONT platforms to rapidly generate reference-quality clinical isolates of AMR priority pathogens and identify potential vaccine targets.

Advances in liquid biopsy technologies and single-cell genomics are transforming cancer detection and our understanding of tumor evolution. In this talk, I will present two parallel efforts leveraging these applications: (1) the development of liquid biopsy approaches for early cancer detection, and (2) the use of long-read single-cell sequencing to study the evolution of breast cancer in solid tissue biopsies.

First, I will discuss our work in developing liquid biopsy methodologies for detecting early-stage breast, prostate, and pancreatic cancers. By integrating circulating cell-free DNA (cfDNA) profiling with epigenomic and mutational analyses, we are identifying molecular signatures that can serve as biomarkers for early disease detection and risk stratification. Using population-based cohorts such as CanPath and the Ontario Health Study, we are refining these approaches to enhance their clinical utility and scalability.

Second, I will highlight our investigations into breast cancer evolution using Oxford Nanopore long-read single-cell sequencing in solid tumor biopsies. This work focuses on characterizing intra-tumoral heterogeneity, clonal selection, and interactions within the tumor microenvironment. By capturing the full complexity of mutational and epigenomic landscapes at the single-cell level, we are uncovering key drivers of tumor progression and therapy resistance. These insights are crucial for improving personalized treatment strategies and understanding the biology of breast cancer in diverse patient populations.

Together, these studies demonstrate the power of nanopore sequencing in both minimally invasive early cancer detection and deep molecular profiling of overlapping solid tumors. By integrating these approaches across large population cohorts and clinical samples, we aim to advance precision oncology and contribute to more effective cancer diagnostics and therapeutics.