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Warren Bach, Seth Cheetham and Josie Gleeson

Lorne Genome 2022 Workshop

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Speaker: Warren Bach, Strategic Relationship Manager ANZ, Oxford Nanopore Technologies

Title: Through the Nanopore – Updates and developments

The DNA and RNA sequencing platform from Oxford Nanopore Technologies has been under rapid development over recent years. Hardware, software and consumables have all undergone significant improvements and diversification. This truly unique and innovative technology enables the application of sequencing in traditional and non-traditional applications from human health to environmental health and everything in between. This presentation will give a short overview of the technology and provide an update on the latest developments and applications, many of which were released at our user group meeting, Nanopore Community Meeting 2021 in November.

 

Speaker: Seth Cheetham, Mater Research Institute-University of Queensland

Title: Single-molecule simultaneous profiling of DNA methylation and DNA-protein interactions with Nanopore-DamID

DNA-protein interactions and cytosine methylation control eukaryotic gene expression. Here, we present an approach to simultaneously detect cytosine methylation and DNA-protein interactions from single molecules, through selective sequencing of adenine-labelled DNA. Applying this approach to LaminB1-associated heterochromatin domains, we identify strict CpG methylation maintenance at transcriptional start sites amid a generalised relaxation of methylation, potentially to prevent ectopic aberrant heterochromatic gene expression. Finally, with Nanopore-DamID we identified allele-specific interactions with CTCF that are associated with X chromosome inactivation.

 

Speaker: Josie Gleeson, Centre for Stem Cell Systems, Department of Anatomy and Physiology, The University of Melbourne, Parkville, VIC, Australia

Title: Accurate expression quantification from nanopore direct RNA sequencing with NanoCount

Sequencing full-length native RNAs using long-read direct RNA sequencing (DRS) has the potential to overcome many limitations of other sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. We developed NanoCount for fast, accurate transcript isoform quantification in DRS and demonstrate it outperforms similar methods. Using synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that DRS accurately quantifies RNA expression and identifies differential expression of genes and isoforms. Our results demonstrate enhanced DRS isoform quantification with NanoCount and establish the ability of DRS to identify biologically relevant differential expression of genes and isoforms.