WYMM Tour: Singapore

Loss-of-function (LoF) variants in the FLG gene are associated with two very common skin diseases: ichthyosis vulgaris (IV) and atopic dermatitis (AD). The FLG gene encodes for filaggrin, is extremely repetitive and difficult to study with most sequencing methods. Therefore, we utilized long reads from two nanopore sequencing strategies: targeted amplicon sequencing and adaptive sampling, to determine sequence variants and intragenic-repeat copy number variants (CNVs) in this 14kb gene.

IV and AD patients with two FLG LoF variants tend to have a more severe disease course, but subsets of compound heterozygous patients display milder phenotypes. The underlying genetic mechanism of this occurrence is unknown. One hypothesis is that two LoF FLG variants could be located in cis, thus altering the predicted gene dosage events in a patient. With nanopore sequencing strategies, we investigated allelic phasing of FLG genetic features in a cohort of patients with predetermined compound heterozygous genotypes and CNVs.

We were able to show that nanopore reads enabled detection and accurate phasing of CNVs and LoF variants into parental alleles, providing in-depth genetic analysis of IV and AD skin diseases. This study highlights the utility of long reads to investigate the genetic contributions of FLG to dry and immunological skin disorders of clinical relevance.

Pharmacogenomics (PGx) holds the promise of delivering personalized medicine, tailoring drug prescriptions to individual genetic profiles. In Thailand, this approach has been integrated into healthcare guidelines, notably for HLA-B genotyping in the context of prescribing allopurinol and carbamazepine. Patients carrying the HLA-B15:02 allele are advised against carbamazepine use, while those with HLA-B58:01 should avoid allopurinol due to heightened risks of adverse drug reactions (ADRs). Current testing methodologies, such as PCR-SSP and PCR-SSOP, are designed to identify a single allele with a binary outcome, simply classifying the HLA-B allele as "positive" or "negative". This binary reporting system overlooks the complexity of genetic interactions that are often influenced by ethnic and racial backgrounds, exemplified by the association of HLA-A31:01 with carbamazepine-induced ADRs in Japanese or Caucasian populations. Consequently, a positive or negative report for HLA-B15:02 does not yield significant clinical value for individuals from these ethnic groups due to the genetic variability and population-specific allele frequencies.

Our study proposes the use of nanopore sequencing technology as a comprehensive solution. This innovative method allows for the reporting of complete HLA-B genotypes, potentially with a single lifetime test. Nanopore sequencing offers the distinct advantage of providing same-day genotyping results. We will share our experiences utilizing nanopore technology to report HLA-B genotypes in Thailand, and the efforts to advocate for its adoption within the Thai healthcare system. Our goal is to demonstrate how nanopore sequencing can be a transformative tool in expanding drug coverage (such as abacavir, dapsone, oxcarbazepine, etc.) and enhancing the precision of pharmacogenomic applications, ultimately paving the way for safer, more effective drug therapy for all.

Long read sequencing is an emerging technology that is expected to provide a more complete reading and understanding of genomes, epigenomes, and transcriptomes. It has the ability to accurately sequence complex repetitive DNA regions or resolve phasing and complex genomic rearrangements. In this talk, we will discuss several clinical research cases in which the application of this technology has informed potential improvements to patient care.

Phased genome assembly traditionally require the use of at least two distinct long-read technologies: ultra-long nanopore simplex reads, and either PacBio HiFi or nanopore duplex reads. However, our research indicates that self-corrected ultra-long nanopore reads alone are adequate for achieving this goal. We have developed a tool called HERRO, which specializes in the error correction of ultra-long reads while taking into account nucleotide variations characteristic of each haplotype. Herro is a two-stage technique that integrates read overlapping with Ai based error correction. It utilizes a blend of convolutional neural networks and self-attention mechanisms within its architecture. Correcting R10.4.1 ultra-long reads, Herro achieves a significant increase in accuracy, reducing the error rate by more than an order of magnitude. By employing only corrected ultra-long reads, Herro demonstrates results in de novo assembly of the human HG002 genome that are on par with or surpass those obtained by methods that combine uncorrected ultra-long reads with either PacBio HiFi or duplex nanopore reads.

Genomic structural variants (SVs) are widespread in human genetic disease genomes. SVs can affect the cell’s growth, development and function by perturbing gene structures and expression regulation. The majority of SVs have not been identified by short-read sequencing because of the complexity and the long span. Nanopore long-read DNA sequencing technologies offer significantly longer read lengths, which will facilitate the detection and analysis of highly rearranged genome alteration. A comprehensive understanding of the structure and distribution of SVs in disease genome will empower the biomedical research community to reveal mechanisms that induce genome variation, identify prognostic signatures of disease and suggest potential targets for novel treatment strategies.

The unveiling of the complete haploid human genome (CHM13) and the successful generation of a high-quality, fully phased diploid genome (HG002) mark significant progress in genetic research. Despite these achievements, existing reference genomes do not fully represent the extensive spectrum of genetic variations across diverse populations, particularly within the Asian demographic, which constitutes over 60% of the global population. Although two fully phased, high-quality genomes exist for individuals of Han Chinese descent, addressing the broader Asian genetic diversity remains a critical gap. In this context, our research aims to propel this initiative forward by meticulously constructing an accurate, gapless, fully phased diploid genome from a male individual with Indian heritage. This initiative is a foundational step toward establishing the Asian pangenome, enhancing our understanding of genetic variations in the Asian population. The intricate process involves advanced sequencing technologies, encompassing assembly, scaffolding, gap filling, rDNA analysis, structural variation (SV) analysis, manual curation, and genome-wide annotation. Our meticulous methodologies and advanced analyses resulted in a detailed portrayal of the Indian genome, including 44 autosomes and 2 sex (XY) chromosomes at the telomere-to-telomere (T2T) level. Utilizing this genome, we conducted comprehensive comparisons with established reference datasets to discern unique genetic characteristics. This comprehensive reference will function as an alternative reference for genomic studies within the South Asian population.

The relationship between structure and their function is the heart of modern cell biology. However, unlike stable protein structures, the studies of flexible RNA structures are still underappreciated due to the technique limitations. With new technologies developed such as high throughput sequencing and the emergence of new structure probing compounds, RNA structure studies have been growing fast recently. However, previous short reads sequencing requires a reverse transcription and PCR (RT-PCR) step which could introduce bias, and this approach would shear each full-length molecule into small fragments. To overcome these problems, we use Nanopore direct RNA sequencing to detect RNA structure, with this approach, we could detect and calculate RNA structure heterogeneity at single molecule level. Briefly, we first use a structure probing compound to treat the RNAs, this compound will introduce the modifications at the single strand area of an RNA molecule, after using Nanopore direct RNA sequencing to detect the introduced modifications, we then could calculate the RNA structure heterogeneity through the modification numbers on each base of an RNA molecule.

Leveraging the effectiveness of Bambu, a tool for transcript discovery and quantification, in conjunction with long-read RNA-seq, we extend its application to single-cell data, aiming to provide precise isoform-level quantification and discovery at the cell-resolution. Single-cell data introduces unique challenges, including the need for de-multiplexing, lower sequencing depth compared to bulk data, and potential interference among diverse cell types. Here, we introduce the beta version of a single-cell-adapted Bambu workflow, offering accuracy, speed, and efficiency. We assess Bambu's performance on both 5' and 3' single-cell data in comparison to bulk data.