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（由申请人提供）：克氏锥虫基因组研究最持久且最显着的障碍是缺乏可接受的参考基因组。有许多因素阻碍了生成高质量、完全组装的克氏锥虫参考基因组的进展。序列，包括 T. cruzi 基因组中的高重复含量 (50%)、被选择用于测序和参考基因组组装的参考菌株 (CL-Brener) 中的极端全基因组杂合性，以及遗传因此，T. cruzi 的当前参考基因组具有数百个间隙和组装崩溃区域，从未完全组装，并且实际上由于参考菌株的杂合性而呈现为两个基因组 -每条染色体都呈现为两个版本，在基因内容、两条染色体序列对齐的序列同一性、甚至成对中两条染色体的长度方面存在显着差异，这些问题极大地削弱了先前和正在进行的全基因组的功效和力量。这方面的测序研究人类疾病的病原体，并且几乎所有涉及审查参考基因组的努力都变得困难且经常无效，我们建议使用最初用于生成它的方法的三种修改来解决当前克氏锥虫参考基因组的问题。首先，我们建议使用单分子长读长（长达 10 kb）测序来缩小间隙并扩大主要使用 Sanger 读长（700 bp）组装原始 CL-Brener 时发生的组装崩溃区域其次，我们将选择纯合且不具有嵌合基因组的 T. cruzi 分离株来简化组装。第三，因为所有 T. cruzi 品系均源自两个祖先、不同的、纯合的非嵌合谱系。 ，我们将为每个“亲本”谱系生成参考序列，以涵盖所有克氏锥虫分离株的遗传内容范围。