Cancer Nanopore Day, Sydney 2023

The characterization of somatic mutations through genome sequencing of tumors has revolutionized cancer research, playing a crucial role in understanding tumorigenesis, tumour heterogeneity and identifying potentially actionable targets. To date, the majority of cancer genome studies have been conducted using short-read sequencing. Long-read sequencing using the Oxford Nanopore Technologies (ONT) enables direct sequencing of DNA allowing simultaneous whole genome sequencing and methylation profiling. In this talk, we will share our experiences of long read whole genome analysis to evaluate several approaches for mutation detection and cytosine methylation profiling.

Widespread genomic aberrations are hallmark of many cancer types. However, the identification of complex driver events such as structural variants (SV) remains challenging. The short-read whole genome sequencing (WGS) has limited ability to handle SVs. Here we performed Oxford Nanopore WGS on tumour samples from distinct cancer types. We developed in-house consensus analysis workflow to identify consensus and somatic SVs. Additionally, we utilized denovo assembly to delve deeper into cancer genome structure and misassembled regions associated with the identified consensus SV calls. This investigation showcases utility of Oxford Nanopore sequencing for comprehensive analysis of SVs and complex cancer genomes.

The ribosomal RNA gene (rDNA) repeat encodes ribosomal RNA (rRNA), central to ribosomes and cellular growth. While inactive rRNA genes possess a closed chromatin state with high DNA methylation, active ones exhibit an open state with reduced methylation. Malignant transformation sees significant rDNA chromatin changes, affecting transcription factor binding and methylation. These alterations drive the anomalous transcription of rDNA genes. We found unexpected methylation patterns in malignant samples, challenging elevated upstream binding factor (UBF) binding trends during malignancy. To address this, we integrated ChIP-seq with ONT, deciphers the concurrent DNA sequence, methylation, and bound proteins across disease stages, shedding light on chromatin structural changes in cancer progression.

We employ innovative Oxford Nanopore Technology direct RNA sequencing (DRS)-based tools, to define isoform-resolved transcriptomes and epitranscriptomes of REH (ETV6-RUNX1) and KOPN-8 (KMT2A-MLLT1) B-cell acute lymphoblastic leukemia (B-ALL) cell lines. We detect multiple incidents of isoform switching in REH and KOPN-8, and prominent differences in the m5C, m6A and pseudouridine patterns. We further reveal differentially translated (and modified) transcripts related to mitochondrial function, cell cycle regulation, DNA binding and repair in both malignant cell lines, implying re-tuned survival and adaptation strategies. Our work highlights the importance of accurate transcriptome and epitranscriptome definition using ONT in cancer biology.

Approximately half of the human genome is composed of mobile DNA sequences. These are a major endogenous source of genetic variation and disease and have historically been difficult to study due to their highly repetitive nature. Oxford Nanopore Technologies (ONT) long-read sequencing solves this problem and is transforming the mobile DNA field. In this talk, I will present ONT data from a range of biological contexts, which altogether depict DNA methylation as the key determinant of mobile DNA expression and cis regulatory activity in somatic cells.

To investigate the genetic basis of cancer and determine its clinical course we developed several novel approaches that leverage the properties of nanopore sequencing. The first area involves the characterization of single cell mutations and cellular engineering of somatic variants. Using nanopore sequencing we have strategies that enable us to detect cancer mutations and gene fusions from mRNA at the resolution of single cells. This method has proven highly efficient and enables one to increase the multi-omic scope of single cell RNA-seq studies. To study the consequences of these mutations, we have developed single cell CRISPR base editor methods to introduce mutations into cells in highly parallel fashion. We use nanopore sequencing to identify the engineered mutations and determine their phenotype derived from gene expression. Finally, we have developed approaches to identify cancer based on cell free DNA methylation and fragmentomic profiles. These approaches leverage the specific properties of nanopore sequencing-based genomics.

Long-read transcriptome sequencing enables full-length transcript discovery and expression level quantification. Coupled with single-cell sequencing, tissue heterogeneity can be profiled at an unprecedented resolution. However, challenges remain in how to analyse this complex data. My research group is developing and applying computational methods for long-read transcriptomics to uncover changes in cancer. Here I will present several examples of past and ongoing work such as JAFFAL - a long-read fusion finder and Flexiplex - a long-read single-cell demultiplexing and search tool, and describe how we use long-reads to improve clonal resolution in single cell data.

TBC

TBC - Work in progress