Finishing multiple genomes in EupathDB using Oxford Nanopore Single Molecule sequencing

项目总结/摘要： 弓形虫是人类重要的条件致病菌，可引起严重的疾病， 发育中的胎儿和艾滋病毒/艾滋病患者。尽管研究界做出了大量的努力来测序， 组装和注释这种生物的多个基因组，这些基因组序列仍然不完整， 到重复和不可克隆的序列。这种知识差距的一个主要原因是， 所使用的技术（第一代和第二代）不能完全解析这些基因座。这妨碍了充分有效地利用 研究社区的数据（托管在EuPathDB生物信息学资源中心; BRC） 因为存在数千个碱基对的缺失和/或未组装的数据。在这里，我们建议重新排序 并为多个T产生从头组装。弓形虫分离株（以及其他两个物种， 比较者）使用第三代测序和基于染色体构象的测序方法， 然后注释它们并将它们集成到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T.刚地（发现它拥有13个，而不是 超过14条染色体），将总基因组组装增加~2 Mb，并对 在T中具有已知作用的基因座处的结构和/或拷贝数变异。弓形虫发病机制拟议 研究响应RFA PA-19-068，“现有数据集的二次分析，用于推进感染性 疾病研究”通过专门使用EuPathDB BRC之外的数据（我们的从头组装和 注释），以提高EuPathDB BRC中数据的实用性（基因表达，注释和 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中的数据来识别我们的新组装体与那些组装体之间的序列差异 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，所提出的研究的成功也促进了组装 团队，包括一名基于染色体构象捕获的测序方法专家（Le Roch） 和序列组装和注释（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).