Virtual Nanopore Day, Australia: Cancer research with nanopore sequencing

Speaker Details:

Warren Bach, Strategic Relationship Manager, Oxford Nanopore Technologies 

Warren joined ONT in 2019 to support researchers, provide updates as the technology develops and communicate its capabilities across Australia, New Zealand and Singapore. Over 20 years’ experience in both sales and product management roles across a range of genomics technologies including PacBio, Fluidigm, Bionano Genomics, 10X Genomics, Affymetrix etc. Prior scientific career started in biotechnology working in Australia and Japan focusing on recombinant protein expression and purification methods development, scale-up and GMP production.

Mike Yarski, Field Applications Specialist, Oxford Nanopore Technologies 

Mike joined ONT in November 2019 as a Field Application Specialist to support researchers in Australia, New Zealand and Singapore by providing updates and trainings as the technology develops. Prior to working for Oxford Nanopore, Mike spent over 10 years working for a leading Australia and New Zealand distributor supporting a range of genomic technologies including Advaita, PacBio, Fluidigm, 10X Genomics, and Affymetrix both as a technical sales specialist as well as an applications specialist. Prior to his commercial roles, Mike completed his PhD at the University of California, Irvine before relocating to Melbourne, Australia where he worked in a few labs as a post-doctoral scientist.

Logan Walker, University of Otago Christchurch

Associate Professor Walker is a Rutherford Discovery Fellow at the University of Otago. His research programme includes developing new methods for evaluating the clinical significance of genetic variants. He is a member of several international groups tasked with improving variant classification, including the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) Splicing Working Group Chair and Expert Panel, the ClinGen Hereditary Breast Ovarian and Pancreatic Cancer Variant Curation Expert Panel and the ClinGen Sequence Variant Interpretation - Splicing Sub-group, and the BRCA Challenge Steering Group.

Long-read assessment of gene transcripts for developing variant classification guidelines

Analysis of ‘naturally occurring’ mRNA splice isoforms for breast cancer susceptibility genes aids in the interpretation of in vitro splicing assays for sequence variant classification. We have been applying long-read nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and PCR-based technologies to catalogue alternative mRNA transcripts from breast and ovarian cancer susceptibility genes. We are using these data to evaluate potential pathogenicity of gene variants located at canonical splice sites and refine the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) variant classification guidelines.

Marjan Naieni, QIMR Berghofer Medical Research Institute

Marjan completed a PhD at Genome Institute of Singapore, A*STAR, Singapore in 2018. She is currently a Research Officer in the Medical Genomics group at QIMR Berghofer Medical Research Institute. She is a bioinformatician whose research focuses on developing and applying computational approaches to study cancer. She has been awarded as an early career researcher from Australian Skin and Skin Cancer Research Centre (ASSC) to conduct a pilot study investigating genomics of melanoma using Oxford Nanopore long read sequencing Technology (ONT).

PromethION long read sequencing to study cancer genomes and epigenomes

Widespread genomic and epigenomic aberrations are a hallmark of many cancer types. Despite their contribution in oncogenesis, identification of complex driver events such as structural variants (SV) in cancer remains challenging. Current short read whole genome sequencing (WGS) technologies have limited ability to handle repetitive regions and SVs. Oxford Nanopore Technologies (ONT) offer improved identification of SVs and epigenomic events. Here, we applied PromethION ONT on tumour samples and cancer cell lines to compare this technology with Illumina short read whole genome sequencing and EPIC array methylation profiling.

Rachel Thijssen Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia

Dr Rachel Thijssen is a Postdoctoral Fellow at the Walter and Eliza Hall Institute of Medical Research. She completed her PhD within the Immuno-Hematology department of the Academic Medical Center (AMC), Amsterdam, the Netherlands before moving to Australia. Her research focuses on unravelling how blood cancer cells evade cell death, particularly elucidating the emergence of resistance in venetoclax treated CLL using state-of-the-art technologies such as single cell sequencing. She is an emerging leader in the field of translational research in cell death and blood cancers.

Jafar S. Jabbari, Australian Genome Research Facility, Melbourne, Vic, Australia

Jafar Jabbari is a molecular biologist and holds a PhD in functional genomics. He is Principal Scientist at AGRF whereby he is responsible for the development of novel and state-of-the art techniques within the- Innovation and Development department. Jafar has a diverse scientific background that includes molecular genetics, genomics and over a decade working with short- and long-read next generation sequencing technologies. Jafar’s current research interests are focused on high throughput single cell full-length isoform sequencing and he is collaborating with multidisciplinary research groups to develop technologies that addresses novel issues and brings together multiple technologies in order to contribute to patient care and advance scientific knowledge.

High Throughput Chronic Lymphocytic Leukemia (CLL) Patient Single Cell Full-length Transcriptome Sequencing on PromethION

Single cell omics, and especially RNA-seq technologies, have developed rapidly in recent years and been instrumental in identifying new cell types, inferring developmental trajectories, and having other applications. These approaches mostly involve sequencing the 3’ or 5’ ends of poly-A RNA on short read platforms. However, additional biological insights can be obtained by adapting these methods to long-read sequencing technologies such as Nanopore to sequence full-length transcripts, thereby studying the possible role of isoforms resulting from alternative splicing. Nevertheless, utilising long-reads for single cell analysis has been hampered by relatively high costs and low accuracy associated with long-read sequencing.
Consequently, we have developed strategies to overcome these obstacles enabling efficient parallel analysis of isoforms from thousands of single cells. In this presentation, we will describe the technologies and their application to profile thousands of PBMC cells from CLL patients and cell lines. These results demonstrate the power of Nanopore sequencing and single cell isoform analysis to obtain better biological insights.

Yuichi Shiraishi, National Cancer Center Research Institute, Japan

Yuichi Shiraishi obtained Ph.D. in statistical science at the Institute of Statistical Mathematics. After that, he worked on developing statistical methods and software for analyzing cancer genome sequence data at RIKEN and the University of Tokyo. Currently, he is engaged in the development of a platform for whole genome and whole transcriptome sequence analysis platform in National Cancer Center.

Systematic characterization of somatic structural variations and mobile element insertions from paired long-read sequencing

Here, we introduce our novel software, nanomonsv (https://github.com/friend1ws/nanomonsv) for detecting somatic structural variations (SVs) using tumor and matched control long-read sequencing data with a single-base resolution. We demonstrate that our approach can detect novel somatic SVs, especially those whose breakpoints are located in repeat regions. Also, we elucidate their in-depth properties such as 5′ truncations, internal inversion, and source sites in the case of LINE1 transductions. Finally, we delineate complex SVs probably evoked by replication mechanisms or telomere crisis by investigating the conjunction of multiple somatic SVs in common supporting reads.

Seth Cheetham, Mater Research Institute-University of Queensland

Dr Seth Cheetham is an NHMRC Early Career Fellow in Professor Geoffrey Faulkner’s Group at Mater Research Institute-UQ. Seth conducted his Honours project with Professor John Mattick at the Institute for Molecular Bioscience at UQ, focussed on differentiating human protein-coding and noncoding RNA transcripts. For his PhD, he joined the group of Professor Andrea Brand at the University of Cambridge to study the functions of long noncoding RNAs (lncRNAs) in neural development and the mechanisms through which they act. Dr Cheetham’s current research focuses on deciphering the functions of repetitive elements in cancer using novel tools including third generation sequencing platforms and DamID-based approaches.

Nanopore sequencing enables comprehensive profiling of transposable element insertions and epigenetic states in cancer.

In cancers, but not healthy tissues LINE-1 “jumping genes” insert throughout the genome, sometimes activating oncogenes and disrupting tumour suppressor genes. While CpG methylation regulates LINE-1 activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of LINE-1s in paired tumour and non-tumour liver as well as healthy tissues. We find pronounced demethylation of LINE-1s in cancer, allele-specific LINE-1 methylation, and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumour-specific LINE-1 insertions.