Accurate Analysis of Nanopore Sequence Data to Drive Crop Improvement

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Accurately determining genome organization and sequence enables efficient genetic improvement of crop species. Long-read sequencing technologies such as Oxford Nanopore Technologies (Nanopore) are essential for capturing structural and sequence variation as well as the complete transcriptome, however, challenges remain, including false mapping, mis-assembly and failure to identify full-length isoforms. Real-time selective sequencing of specific regions of small genomes using Nanopore has been demonstrated (Loose et al, 2016, Nature Methods), but this cutting-edge technology needs adapting for larger genome crop species. We are working to improve long read sequencing in barley, with key target traits being yield, flowering time and nutrient water use efficiency.Objectives The aim of this interdisciplinary project is to develop computational methods to enhance the accuracy of Nanopore sequencing analysis, and use these methods to increase our understanding of the barley genome, variation across barley lines, and transcript-specific expression. This will provide key information to underlie a mechanistic understanding of barley traits. MethodologyWe have developed a novel computational approach for PacBio Iso-seq analysis (Zhang et al, Genome Biology, 2022), which can effectively identify and remove inaccurate transcripts in PacBio Iso-seq data. We will test and improve this approach on Nanopore sequencing data to annotate our new barley high-quality genome assembly and also improve understanding of the accuracy, coverage, and dynamic range of Nanopore RNA sequencing with respect to quantitative analysis of mRNA expression. Furthermore, we will improve the selective sequencing technology for Amaranth using the nanopore "Read-Until" technology.