WGGC Webinars | Dr. Sebastian Scharf | Microbiomes & host–pathogen interactions with ONT

Title: Investigating microbiomes and host–pathogen interactions ONT long-read sequencing We have developed a metagenomics pipeline for long-read sequencing that enables the simultaneous profiling of bacterial, fungal, archaeal, eukaryotic, and viral components, as well as antibiotic resistance genes. When applied to a longitudinal cohort of patients who underwent allogeneic hematopoietic stem cell transplantation (n = 31), this approach revealed distinct microbiome clusters prior to transplantation, high interindividual variability, and dynamic shifts in microbial composition, including significant contributions from viruses and fungi. Comprehensive characterization of complex microbiomes requires robust sequencing and analysis approaches. Multiplex long-read DNA sequencing using Oxford Nanopore Technologie (ONT) with specific barcodes enables higher throughput and lower costs but is susceptible to barcode misassignment (crosstalk), particularly in samples with low input DNA. We systematically evaluated barcode crosstalk across three ONT library preparation protocols and applied an optimized protocol for ONT multiplexing.