WYMM Tour: Bangkok

Long-read sequencing has emerged as a pivotal technology in clinical optimization, particularly for applications where structural variations and phasing are critical. The integration of cost-effective next-generation sequencing (NGS) pipelines into clinical practice is essential for sustainable implementation at a national scale. Establishing research and excellence centers across the Ministry of Public Health will be key to ensuring access to high-quality sequencing services and advancing sequencing technology in Thailand. This approach not only enhances healthcare quality but also positions Thailand as a leader in genomic research and technology in the region.

Accurate and rapid diagnostics of pathogens have important implications for navigating the treatment of infectious diseases and preventing possible outbreaks. However, accurate microbiological diagnosis involves numerous laboratory procedures including culture, biochemical tests, serology tests, and molecular assays, often the pathogen is not identified in time to guide clinical management. Moreover, the limitation of culture for some microbes diminished the ability to identify pathogens. Additionally, false positives due to blood culture contamination are common, leading to complications in results interpretation from detecting microbial contamination rather than the true infection. NGS with a culture-free approach could enhance the ability of the laboratory to identify causative pathogens. With NGS becoming cost-effective and less time-consuming, it has high potential in routine diagnostics and conducting infectious etiologies in a large cohort. Recently, we have developed ONT sequencing with a pooling method for identifying causative agents of sepsis and fever of unknown origin (FUO). In Thailand, sepsis affects approximately 175,000 adults each year with a mortality rate of 25%. There is a limited epidemiological report of sepsis, especially the uncommon pathogens. FUO or pyrexia of unknown origin (PUO) can be caused by many diseases: infection (account for 17–35% of cases), inflammatory, neoplastic, and miscellaneous. Although the overall mortality of FUO is low, patient management should be supportive until the cause has been determined. Due to various causes and limited detection methods, nearly half of the FUO cases remain undiagnosed. Numerous infectious agents; including bacteria, viruses, fungi, and parasites, account for sepsis and FUO in Thailand. Our newly developed culture-free NGS-based assay using ONT and pooling methods has been successfully used to detect pathogenic bacteria and fungi in clinical research samples derived from sepsis and FUO patients. Its application in a large cohort etiologies study has been applied and evaluated to support the plans for nationwide studies.

Preimplantation genetic testing for aneuploidy (PGT-A) typically involves trophectoderm biopsy and genetic analysis using next-generation sequencing (NGS). Traditional NGS-based PGT-A often requires several hours for library preparation, sequencing, and data analysis. This presentation will describe a same-day PGT-A method that leverages rapid library preparation techniques and real-time data analysis pipelines. To validate this research approach, a small cohort of biopsy samples from embryos with previously characterized copy number variations (CNVs) were analyzed. We will present data on the workflow and flexibility of Oxford Nanopore Technologies (ONT) in PGT-A testing, demonstrating its potential to provide reliable and timely genetic information for embryo selection.

There are ~200 children in high dependency neonatal acute care in New Zealand at any one time, requiring a scalable distributed solution for acute care genomics. We have established an expandable acute care clinical pipeline based around the PromethION2 solo system with connection to Fabric GEM™. In the establishment phase, we have performed benchmarking using GA4GH benchmarking tools and Genome in a Bottle HG002 - HG007. Evaluating ~3.3x106 truth SNVs and ~500x103 INDELS at read depths of between 24-42X coverage identified SNV recalls = 0.992 ± 0.001, precision = 0.997 ± 0.0006, and F1 = 0.995 ± 0.0008 over a minimum of two runs completed by different technicians and analysts. INDEL identification approached recalls = 0.838 ± 0.043, precision = 0.922 ± 0.019, and F1 = 0.874 ± 0.032 over the same runs. Subsequent analyses indicated that the observed variation in recall, precision and F1 was largely limited to correct copies of falsely duplicated regions and areas of collapsed errors with clusters of CHM13 hets in GRCh38. Rarefaction analyses up to 80X coverage identified that SNV identification plateaus at ~20X coverage, while INDEL identification plateaus at ~40X coverage. Analyses of samples from Coriell CNVPANEL01 demonstrated that large scale genomic variations can be reliably detected after ~2M reads, equivalent to ~2hr sequencing time. Application of the pipeline in acute care genomic diagnosis is ongoing. We present the preliminary results from the pipeline validation phase, performed in parallel with established International accredited facilities available to New Zealand’s clinicians. We also present some data demonstrating the flexibility of the data for analysis of unknowns because knowing is not enough.

Recent advancements in next-generation sequencing (NGS) have profoundly transformed the landscape of rare disease diagnostics, offering unprecedented precision and speed. NGS technology enables comprehensive analysis of genetic material, identifying single nucleotide variations, insertions, deletions, and copy number variations with high accuracy. This is particularly critical for diagnosing rare diseases, which often involve complex genetic anomalies. A notable advancement is the emergence of long-read sequencing technologies, which provide more comprehensive and accurate genetic insights by capturing larger and more complex genomic regions that are often missed by traditional short-read methods. This has significantly enhanced the ability to identify structural variants and repeat expansions associated with many rare diseases, improving diagnostic accuracy and patient outcomes. In this talk, I will share our experiences in the study of rare disease using long read sequencing. Continued innovation and collaboration in this field promise further improvements in understanding and managing rare genetic disorders.

Rare diseases, marked by their low prevalence and elusive genetic origins, have posed formidable diagnostic hurdles. The advent of exome and short-read genome sequencing has indeed transformed rare disease diagnosis, illuminating pathogenic variants. However, a subset of cases persisted unresolved due to challenges in deciphering intricate structural variations. This void has spurred the emergence of long-read genome sequencing.

In this context, we present our experiences harnessing long-read sequencing to identify novel disease genes, particularly those entailing repeat expansions. We further explore its potential utility in diagnosing disorders of cases unsolved by exome and short-read genome sequencing; and in those attributed to difficult genes. Moreover, we delve into its potential application for critically ill patients with a suspected genetic disease, for whom timeliness is paramount.

The profound impact of long-read sequencing on untangling the genetic basis of rare diseases bears immense potential. This advancement promises enhanced patient outcomes and a deeper comprehension of these conditions.

Long-read sequencing clinical research, powered by nanopore technology, has the potential to transform healthcare with its real-time sequencing capabilities, rapid turnaround times, and high-resolution data. This approach delivers groundbreaking advancements across multiple research fields, including pharmacogenomics, cancer, non-communicable diseases, and infectious diseases. This talk highlights the transformative potential of long-read sequencing clinical research in each of these categories, bridging research and service to revolutionize healthcare in Thailand.

Long-read sequencing technologies have emerged as powerful tools for comprehensive genomic analyses, particularly in complex variation of the genome. This presentation will explore findings from the Genomics Thailand cohort, focusing on germline mutations in Thai cancer patients with breast-ovarian and colorectal cancers. We discuss the utility of long-read sequencing in addressing limitations of traditional short-read approaches, including its potential to detect large genomic rearrangements, such as copy number variants (CNVs), and its ability to resolve double pathogenic variants. Key insights highlight the unique mutation spectrum in the Thai population, providing valuable context for precision medicine efforts. Challenges encountered include the accurate detection and interpretation of CNVs and SNVs in highly repetitive or structurally complex regions and the identification of overlapping pathogenic variants. These issues are particularly critical for clinical implication of genomic studies. By leveraging the high-resolution capabilities of long-read sequencing, this study demonstrates its promise in elucidating the genomic architecture of hereditary cancers in Thai populations, ultimately contributing to improved diagnostics, risk stratification, and personalized cancer care in Thailand and beyond.