NCM 2023 Singapore

Nanopore sequencing technology has democratized DNA and RNA sequencing and enables more laboratories and classrooms to engage in genome sequencing. In Thailand, a middle-income nation, Oxford Nanopore Technologies has empowered many institutions to establish affordable long-read sequencing facilities. Siriraj Long-read Lab (Si-LoL) at the Faculty of Medicine Siriraj Hospital is leveraging the technology across various applications. Here we will present on our extensive collaborations with local, international, academic, and private partners. Through these cooperative efforts, we've implemented an extensive variety of distinct nanopore sequencing applications across Thailand, demonstrating the far-reaching utility of this technology.

Abstract coming soon

X-linked genetic disorders tend to affect females less severely than males due to the presence of a second X chromosome that does not carry the deleterious variant. However, the phenotypic expression in females is very variable, which may be explained by which allele is silenced through the dosage compensation mechanism of X chromosome inactivation. Skewed X inactivation, where one allele is preferentially silenced over the other, can significantly impact the severity and penetrance of X-linked mutations in carrier females. When preventative treatment is available, accurate measurement of skewed X inactivation is crucial for predicting disease susceptibility in these individuals. We describe a novel approach using nanopore sequencing to accurately quantify skewed X inactivation in females. By phasing sequence variants and methylation patterns, this single assay detects the disease variant, the skew, and its directionality for any patient, contrary to previous methods that require trio information, polymorphism in certain repeats, and additional genetic testing. Our study includes a cohort of X-linked mutation carrier females affected by various diseases impacting the retina. As retinal DNA cannot be readily obtained, we instead determine the skew from three peripheral tissues (blood, saliva, and buccal swab), and correlated it with disease severity and phenotypic outcomes. By using adaptive sampling on the PromethION to enrich for X-chromosome reads, we could multiplex of samples and improve cost-efficiency. Our method of assessing skewed X inactivation is applicable to all nanopore genomic datasets, providing insights into disease risk and severity and aiding in the development of individualised strategies for X-linked mutation carrier females.

RNA modifications such as m6A methylation form an additional layer of complexity in the transcriptome. Nanopore direct RNA sequencing can capture this information in the raw current signal for each RNA molecule, enabling the detection of RNA modifications using supervised machine learning. In this presentation I will introduce m6Anet, a neural-network-based method that leverages the multiple instance learning framework to obtain read-level and site-level m6A modification probabilities. The m6Anet method outperforms existing computational methods, shows similar accuracy as experimental approaches, captures the underlying read-level stoichiometry, and generalizes with high accuracy to different cell lines and species. Finally, I will provide an update on m6A identification using RNA004 with m6Anet.

Epigenomic aberration is one of the hallmarks of cancer genomes. Global hypomethylation, aberrant hypermethylation at transcriptional regulatory regions (e.g. promoters, enhancers) and loss of imprinting have been widely revealed by short-read sequencing and methylation microarrays, but it can be difficult to assess the detailed modification status on a long, continuous read. Here we evaluated the accuracy and feasibility of native DNA sequencing using PromethION from Oxford Nanopore Technologies to detect a long-ranged and haplotype-resolved methylation level in the cancer genome. In addition, we propose the importance of integrated analysis of cytosine modifications to consider the dynamics of 5-methylcytosine and its oxidation derivatives.

Long-read sequencing based de novo and somatic structural variant (SV) discovery remains challenging, necessitating genomic comparison between samples. Here, SVision-pro visually represents genome-to-genome-level sequencing differences and comparatively discovers SV between genomes by a neural-network-based instance segmentation framework without prerequisite of SV inference models. SVision-pro outperforms state-of-the-art approaches, particularly in resolving complex SV (CSV), with low Mendelian error rates and high sensitivity of low-frequency SVs. Moreover, SVision-pro successfully discoveries 26 high-quality de novo SVs in six family datasets and retrieves eight somatic CSVs in normal-tumor-paired samples.

Abstract coming soon

Liquid biopsy using cell-free DNA (cfDNA) in plasma has provided a non-invasive approach for prenatal and cancer testing. However, the analyses of cfDNA have been focused on short DNA molecules (e.g., ≤ 600 bp). Using long-read sequencing technologies, our group revealed the presence of long cfDNA in plasma samples from healthy subjects, pregnant women, and cancer patients. We characterized the fragmentomic and epigenetic features of long cfDNA - taking advantage of the ability of long-read sequencing to directly analyze the methylation patterns of multiple CpG sites in each long cfDNA molecule, we developed an approach to determine the tissue-of-origin of individual long cfDNA molecules. Potential clinical applications of the analysis of long cfDNA, such as the detection of preeclampsia, the prenatal testing of monogenic diseases, and the detection of liver cancer, have been demonstrated in our recent proof-of-concept studies. These studies marked the beginning of a new era in liquid biopsy.

Long-read transcriptome sequencing enables full-length transcript discovery and expression level quantification. Coupled with high-throughput single-cell sequencing, this allows tissue heterogeneity to be profiled at an unprecedented resolution. This is particularly beneficial when applied to cancer, as clone specific variants and isoforms missed by short-read sequencing can be identified. However, analysing long-read data presents some challenges. For example, it is complex to efficiently demultiplex hundreds to thousands of cellular barcodes, in tens of millions of reads. Particularly when the reads have a high number of errors, including insertions and deletions, and some reads are chimeric, containing multiple cellular barcodes and transcripts. To address these challenges, we have developed Flexiplex, a versatile, efficient, and noise-tolerant tool for sequence searching and demultiplexing. Our benchmarking shows that Flexiplex provides an excellent balance between accuracy and computational performance, compared to existing single cell demultiplexing methods. Furthermore, we demonstrate its utility to genotype individual cells to trace clonal variation in cancer and enhance fusion gene detection when used alongside our recently developed long read fusion finder, JAFFAL. Flexiplex is lightweight, fast, and user-friendly in terms of installation and operation. The source code, alongside pre-built binary executables for Linux and macOS, can be accessed at https://davidsongroup.github.io/flexiplex/

Yellow fever virus (YFV) is the prototype species of the Flavivirus genus and has raged across Africa and the Americas for centuries. Fortunately, the extensive implementation of a live attenuated vaccine (YFV-17D) successfully dampened its epidemics, but as an RNA virus, YFV mutates rapidly in response to selective pressure. Utilizing Oxford Nanopore’s Direct RNA Sequencing Kit and a YFV-17D sequence-specific reverse transcription adaptor, the full-length YFV-17D reads with ~370X depth were captured from infected human hepatoma cells, among which only 4% of the reads mapped to the host genome. By using Direct RNA-Seq, we were able to check for genetic variations and look for compensatory mutations that would otherwise be impossible to detect. Soon this workflow will be employed for our upcoming clinical trial involving YFV-17D intrahost evolution in flavivirus naïve individuals and those primed with a Japanese encephalitis virus (JEV) vaccine (NCT05568953). Investigation of these mutations can shed light on their adaptation in human hosts with distinct serological backgrounds and inspire the surveillance of live attenuated vaccine safety, the development of potential differential vaccination regimes, as well as therapeutics.

Abstract coming soon

Abstract coming soon

Drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance worldwide and continues to be a public health threat. Annually, half a million people fall ill with DR-TB globally, and only 1 in 3 have access to diagnosis and treatment. Targeted next-generation sequencing (tNGS) direct from clinical specimens has the potential to transform management of DR-TB by providing rapid and comprehensive drug resistance testing to inform timely treatment, including new shortened drug regimens. Here we will present our findings from the recently completed clinical evaluation of the Oxford Nanopore TB Drug Resistance end-to-end targeted next-generation sequencing solution. This evaluation is part of the Unitaid-funded Seq&Treat grant, designed to generate evidence for WHO policy and catalyze country capacity for the use of targeted next-generation sequencing for DR-TB. We will also present the next steps for country uptake and programmatic implementation of this tNGS workflow.

Abstract coming soon

Every second counts between detection and response. The total cost of an outbreak grows exponentially from time of detection, making identification critical at emergence or early stages of spread. Cambodia is a hotspot of emerging and endemic viruses and the Virology Unit at Institut Pasteur du Cambodge has been performing One Health surveillance at the human-livestock-wildlife interface for 25 years. Improving our ability to monitor multiple pathogens at high-risk interfaces using Oxford Nanopore technology built up during the COVID-19 pandemic sequencing will facilitate response, help develop key biosecurity practices/guidelines to reduce risk, and enable Early Warning. Avian influenza virus (AIV) is causing major global challenges, including spillover into mammalian species and occasionally into humans. Therefore, it is critical to quickly sequence AIV strains to assess risk. We have successfully employed a rapid, integrated barcode, multi-segment protocol for AIV with field-forward bioinformatics to perform surveillance in animal and environmental samples from live bird markets and from human AIV cases in Cambodia. In addition, benchmarking R9 versus R10 chemistry for AIV reveals significant improvement in speed, coverage, and accuracy – with major reductions in indel rates in vitally important low-complexity regions of the virus such as the multibasic cleavage site. Taken together, nanopore sequencing provides a fast and accurate way to quickly respond and assess AIV both in the laboratory and the field, in both endemic and emerging environments.

RNA structures are important in regulating almost every step of an RNA’s lifecycle. While high-throughput structure probing typically involves identifying chemical modifications along an RNA by reverse transcription and deep sequencing, recent developments in direct RNA sequencing enable us to detect RNA structure modifications directly using nanopores. In this talk, we describe our strategy to identify structure modifications on long RNA molecules to identify isoform-specific RNA structures in human ES and SARS-CoV-2 transcriptomes. As direct RNA sequencing allows us to obtain structure signals at a single-molecule level, we will also describe our efforts in studying single-molecule structural heterogeneity using direct RNA sequencing.

Myrtle rust is a fungal disease that is spreading worldwide, threatening the biodiversity of Myrtaceae plants. Many plants will have poor or no defence against this highly infectious, mutating, and rapidly spreading fungus. The extent of post-transcriptional modifications is largely unexplored in pathogen-plant interactions but may play key roles in transcript function and stability during pathogen attacks. We investigated Austropuccinia psidii (myrtle rust) and Syzygium jambos (rose apple) before and after myrtle rust infection, by direct RNA sequencing with the Oxford Nanopore MinION platform. Direct RNA sequencing revealed differential gene expression, alternative splicing, and what dynamic, reversible modifications are made to the RNA, such as methylation. Our research goal is to increase the understanding of pathogen-plant interactions and determine if alternative splicing and RNA modifications play signification roles in infection or plant immunity.

Widespread genomic aberrations are a hallmark of many cancer types. Despite their contribution to oncogenesis, the identification of complex driver events such as structural variants (SV) in cancer remains challenging. Short-read whole-genome sequencing (WGS) technologies have limited ability to handle SVs and complex genomes. Here we performed Oxford Nanopore WGS on tumour samples from childhood cancers and oesophageal adenocarcinoma. We developed an in-house consensus analysis workflow to identify consensus and somatic structural variations. Additionally, we utilized de novo assembly to delve deeper into cancer genome structure and misassembled regions associated with the identified consensus SV calls. The de novo assembly approach effectively covered up to 84% of the genome fraction based on the hg38 reference genome. By comparing our findings with available Illumina short-read WGS data, we discovered novel or improved genomic events using Oxford Nanopore sequencing. The long-read sequencing analysis uncovered multiple complex genomic events, providing higher-resolution insights into cancer-driver genes such as ERBB4, MAP2, IDH1, CASP8, BMPR2 and GATA3. Furthermore, we identified actionable variants such as KIAA1549-BRAF fusion genes with therapeutic implications. This investigation showcases the utility of Oxford Nanopore sequencing for comprehensive analysis of structural variations and complex cancer genomes. Our results contribute valuable insights into the field of nanopore sequencing analysis and shed light on the identification of novel driver events, thereby enhancing our understanding of tumorigenesis and offering potential avenues for therapeutic intervention

Large-scale projects developing perpetual community resources play an increasingly important role in biomedical research and precision medicine. The National Centre for Indigenous Genomics (NCIG), under the Indigenous governance backed by Australian federal statutory powers, establishes genomic reference resources and biospecimen collection from Indigenous Australians. NCIG is an example of a community resource built to enable broad-scale representation of ancestrally diverse populations for equitable benefits of genomics for all humans. NCIG has established deeply trusted relationships with four Indigenous communities since 2015, whereby 684 individuals have donated their genomic data and biological samples to the growing NCIG collection. We have begun generating telomere-to-telomere reference genomes for these four communities leveraging Oxford Nanopore technology and other sequencing platforms. Indigenous reference genomes reveal >2 Mb of new sequences. Similarly, whole-genome sequence data from 163 individuals using Illumina short reads and nanopore long reads show distinct genetic diversity missing from global resources. Of 17 million short variants, ~22% of variants are found only in Indigenous Australians and between 10 - 31% of variants are found only in a single community due to the prolonged isolation from each other and global populations. The unique genetic diversity of Indigenous Australia warrants modifications to the genomic analysis and interpretation workflows to improve research and health outcomes. NCIG continues to expand its genomic reference resources by including more communities and integrating these resources into clinical services and healthcare through national networks.

Genomics Thailand is the first national initiative that puts 16 different research institutes and funding agencies to work under one project. By utilizing the opportunity of various patient’s need for genomic sequencing such as rare diseases, cancer and pharmacogenomics, the project will generate population-level data of 50,000 whole genomes of Thai people. Meanwhile, actionable genome variants that have been identified and systemically interpreted will be reported back to the patients and clinicians. With the mission to integrate genome technology into the national health system, the project also aims to overcome challenges related to clinical implementation, workforce development, data privacy, and ethical considerations while leveraging collaborations with research institutions and industry partners both domestically and internationally. The talk sheds light on the current status and its transformative impact of integrating genomics into the national health system, paving the way for improved diagnosis, treatment, and prevention of diseases, ultimately leading to better health outcomes for the people of Thailand.

Animal agriculture has the amazing ability to turn low quality forages into high quality products for human consumption. Continued expansion of the global population puts increased pressure on agricultural systems to achieve more with less. With nanopore sequencing, the Bovine Long Read Consortium is exploring the dark genome regions, where short-read sequencing technologies cannot be used. This new era of long-read data is allowing us to access new sources of genetic variation, providing promising targets for selection. However, animal agriculture also comes at a cost as methane is emitted as a by-product of digestion. We are using nanopore reads to capture microbiome variation and use that information to identify naturally low methane emitting animals. By combining these different aspects, we look into a future where we can feed the world, without hurting it.

Abstract coming soon

Understanding the mechanism behind human diseases with an established heritable component is the next frontier in personalized medicine. Furthermore, understanding how these mechanisms may vary across ethnicities is crucial for accurate genetic risk prediction in the clinic. To respond to these challenges, we are leveraging Oxford Nanopore technology to discover germline and mosaic variation at scale and with accuracy. Our latest release on Sniffles and Spectra includes novel methods to improve detection of structural and copy number variation across hundreds of individuals. Furthermore, Sniffles2 solves the problem of family- to population-level SV calling to produce fully genotyped VCF files by introducing a gVCF file concept. Across 11 probands, we accurately identified causative SVs around MECP2, including highly complex alleles with three overlapping SVs. Sniffles2 also enables the detection of mosaic SVs in bulk long-read data. As a result, we successfully identified multiple mosaic SVs across a multiple-system-atrophy patient brain. The identified SV showed a remarkable diversity within the cingulate cortex, impacting both genes involved in neuron function and repetitive elements. In my presentation I will outline the novel updates on Sniffles and Spectra to further improve the detection of variants utilizing nanopore sequencing. These updates include improved tumour-normal comparisons, mosaic SV discovery and improvements to population sequencing. Overall, the continuous advancement of our methods coupled with the scale of Oxford Nanopore sequencing at Baylor College of Medicine and worldwide are the steppingstones to improve disease discovery and personalized medicine.

Abstract coming soon