Generation of reference genomes for Trypanosoma cruzi using PacBio sequencing

使用 PacBio 测序生成克氏锥虫参考基因组

• 批准号: 9251753
• 负责人: Rick L Tarleton
• 金额: $ 7.5万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9251753
• 关键词: Address; Americas; Biological Phenomena; Blood; Cells; Chagas Disease; Chromosomes; Communities; Complex; Consensus; Data; Dideoxy Chain Termination DNA Sequencing; Diploidy; Epidemiology; Etiology; Evolution; Future; Gene Duplication; Gene Family; Generations; Genes; Genetic; Genome; Genome Mappings; Genomic DNA; Genomics; Goals; Grant; Heterozygote; Hybrids; Immune Evasion; Institutes; Ions; Karyotype; Laboratories; Length; Libraries; Maintenance; Manuals; Maps; Membrane Proteins; Modification; Molecular Weight; Mosaicism; Nature; Outcome; PPBP gene; Parasites; Pharmacotherapy; Prevention; Protons; Structure; Technology; Testing; Trypanosoma cruzi; Validation; base; duplicate genes; genome sequencing; genome-wide; human disease; improved; interest; next generation sequencing; programs; public health relevance; reference genome; single molecule; virtual; whole genome

 DESCRIPTION (provided by applicant): The most persistent and significant hindrance to genomic studies of Trypanosoma cruzi is the lack of an acceptable reference genome. There are a number of factors that have impeded progress in generating a high quality, fully assembled T. cruzi reference sequence, including high repeat content (50%) in the T. cruzi genome, extreme genome-wide heterozygosity in the reference strain (CL-Brener) that was chosen for sequencing and reference genome assembly, and genetic mosaicism in the reference strain. Thus, the current reference genome for T. cruzi has hundreds of gaps and regions of assembly collapse, has never been fully assembled and is, in fact, presented as two genomes due to the heterozygosity of the reference strain -each chromosome is presented as two versions differing substantially in terms of gene content, sequence identity where the two chromosome sequences align, and even lengths of the two chromosomes in a pair. These issues have greatly muted the efficacy and power of previous and ongoing whole genome sequencing studies in this causative agent of human disease, and have made virtually any endeavor involving the need for scrutinizing the reference genome difficult and frequently unproductive. We propose to tackle the issues with the current T. cruzi reference genome using three modifications of the approach originally used to generate it. First, we propose to use single-molecule, long-read (up to 10 kb) sequencing to close gaps and expand regions of assembly collapse that occurred when primarily Sanger reads (700 bp) were used to assemble the original CL-Brener reference genome. Second, we will select T. cruzi isolates for sequencing that are homozygous and do not have mosaic genomes to simplify the assemblies. Third, because all T. cruzi lines are derived from two ancestral, divergent, homozygous, non-mosaic lineages, we will generate reference sequences to each "parental" lineage in order to cover the scope of genetic content in all T. cruzi isolates.

 描述(申请人提供)：克氏锥虫基因组研究最持久和最严重的障碍是缺乏可接受的参考基因组。有许多因素阻碍了产生高质量、完全组装的克氏锥虫参考序列的进展，包括克氏锥虫基因组中高重复含量(50%)，被选择用于测序和参考基因组组装的参考菌株(CL-Brener)的极端全基因组杂合性，以及参考菌株中的遗传嵌合体。因此，目前克氏锥虫的参考基因组有数百个间隙和组装崩溃的区域，从未被完全组装，事实上，由于参考菌株的杂合性，每条染色体被呈现为两个版本，在基因含量、两条染色体序列比对的序列同一性，甚至一对中两条染色体的长度方面都有很大的不同。这些问题极大地削弱了以前和正在进行的全基因组测序研究在这种人类疾病病原体中的有效性和威力，并使涉及审查参考基因组的需要的几乎任何努力都变得困难，而且往往是徒劳的。我们建议使用最初用于生成T.ruzi参考基因组的方法的三个修改来解决当前T.ruzi参考基因组的问题。首先，我们建议使用单分子，长阅读(高达10kb)测序来填补缺口，并扩大组装崩溃的区域，当主要使用Sanger Reads(700bp)来组装原始CL-Brener参考基因组时。其次，我们将选择纯合且没有马赛克基因组的克氏锥虫分离株进行测序，以简化组装。第三，由于所有的克氏锥虫株系都来自两个祖先的、分化的、纯合子的、非镶嵌的谱系，我们将生成每个“亲本”谱系的参考序列，以涵盖所有克氏锥虫分离物的遗传内容范围。