Finishing multiple genomes in EupathDB using Oxford Nanopore Single Molecule sequencing

项目摘要/摘要： 弓形虫是一种重要的人类条件致病菌，可引起严重的人类疾病。 发育中的胎儿和艾滋病毒/艾滋病患者。尽管研究界做出了广泛的努力来测序， 为这个生物体组装和注释多个基因组，这些基因组序列仍然不完整 到重复和不可克隆的序列。造成这种知识差距的一个主要原因是测序 使用的技术(第一代和第二代)不能完全解决这些区域。这妨碍了充分有效地使用 由研究界提供的数据(托管在EuPathDB生物信息学资源中心；BRC) 因为有数千个碱基对丢失和/或未组装的数据。在这里，我们建议重新排序 并为多个弓形虫分离物(以及作为 比较器)使用第三代测序和基于染色体构象的测序方法， 然后对其进行注释并将其集成到EuPathDB BRC中。我们的初步数据表明， 这种方法，我们已经使用它来修改弓形虫的核型(发现它含有13，而不是 超过14条染色体)，将总基因组组装增加~2Mb，并执行全基因组分析 基因座的结构和/或拷贝数变异在弓形虫致病中具有已知的作用。建议数 对RFA PA-19-068的研究作出了响应，该文件名为“对现有数据集进行二次分析以提高感染性” 通过专门使用EuPathDB BRC之外的数据(我们的新装配和 注释)，以提高EuPathDB BRC内数据的可用性(基因表达、注释和 例如，蛋白质组学数据)。此外，分析管道将依赖于使用现有的基因组序列 EuPathDB BRC中的数据，以识别我们的新程序集与其他程序集之间的序列差异 由BRC主办。除了PI在基因组测序和多拷贝基因座功能方面的专业知识外 编码发病机制决定因素，拟议的研究的成功也是由组装 团队，包括一名基于染色体构象捕获的测序方法专家(Le Roch) 以及序列组装和注释(Lorenzi)。

PROJECT SUMMARY/ABSTRACT: Toxoplasma gondii is an important opportunistic pathogen of humans where it can cause severe disease in the developing fetus and those with HIV/AIDS. Despite extensive efforts by the research community to sequence, assemble and annotate multiple genomes for this organism, these genome sequences remain incomplete due to repetitive and uncloneable sequence. A major reason for this knowledge gap is that the sequencing technologies used (1st and 2nd generation) cannot fully resolve these loci. This prevents fully effective use of the data (which is hosted on the EuPathDB Bioinformatics Resource Center; BRC) by the research community since there are thousands of base pairs of missing and/or unassembled data. Here we propose to resequence and generate de novo assemblies for multiple T. gondii isolates (as well as two other species that serve as comparators) using 3rd generation sequencing and Chromosome conformation-based sequencing approaches, and then annotate them and integrate them into EuPathDB BRC. Our preliminary data show the feasibility of this approach where we have used it to revise the karyotype for T. gondii (discovering that it harbors 13, rather than 14, chromosomes), increase the total genome assembly by ~2 Mb, and perform genome-wide analyses of structural and/or copy number variation at loci with a known role in T. gondii pathogenesis. The proposed studies are responsive to RFA PA-19-068, “Secondary Analysis of Existing Datasets for Advancing Infectious Disease Research” by specifically using data outside of the EuPathDB BRC (our de novo assemblies and annotations) to improve the utility of data within the EuPathDB BRC (gene expression, annotation and proteomics data, for example). Moreover the analysis pipeline will rely on using the existing genome sequence data within the EuPathDB BRC to identify sequence differences between our new assemblies and those hosted by the BRC. In addition to the expertise of the PI in genome sequencing and function of multicopy loci encoding pathogenesis determinants, the success of the proposed studies is also facilitated by the assembled team, including an expert in Chromosome Conformation Capture-based sequencing approaches (Le Roch) and sequence assembly and annotation (Lorenzi).