Accurate Analysis of Nanopore Sequence Data to Drive Crop Improvement

准确地确定基因组的组织和序列，可以有效地进行作物品种的遗传改良。像牛津纳米孔技术（Nanopore technologies）这样的长读测序技术对于捕获结构和序列变异以及完整的转录组是必不可少的，然而，挑战仍然存在，包括错误的定位、错误的组装和无法识别全长同种异构体。利用纳米孔对小基因组的特定区域进行实时选择性测序已经得到证实（Loose等人，2016,Nature Methods），但这种尖端技术需要适应更大的基因组作物物种。我们正在努力改进大麦的长读序列，主要目标性状是产量、开花时间和养分水分利用效率。这个跨学科项目的目的是开发计算方法来提高纳米孔测序分析的准确性，并使用这些方法来增加我们对大麦基因组、大麦系间变异和转录特异性表达的理解。这将为大麦性状的机理理解提供关键信息。我们开发了一种新的PacBio Iso-seq分析计算方法（Zhang等人，Genome Biology, 2022），该方法可以有效地识别和去除PacBio Iso-seq数据中不准确的转录本。我们将在纳米孔测序数据上测试和改进这种方法，以注释我们新的大麦高质量基因组组装，并提高对纳米孔RNA测序在mRNA表达定量分析方面的准确性、覆盖范围和动态范围的理解。此外，我们将利用纳米孔“Read-Until”技术改进苋菜选择性测序技术。

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.