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: 9101686
• 负责人: TODD A MINNING
• 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 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9101686
• 关键词: Address; Americas; Biological Phenomena; Blood; Cells; Chagas Disease; Chromosomes; Communities; Complex; Consensus; Data; Dideoxy Chain Termination DNA Sequencing; Diploidy; Epidemiology; Evolution; Future; Gene Family; Generations; Genes; Genetic; Genome; Genome Mappings; Genomic DNA; Genomics; Goals; Grant; Heterozygote; Hybrids; Immune; Institutes; Ions; Karyotype; Laboratories; Length; Libraries; Maintenance; Manuals; Maps; Membrane Proteins; Modification; Molecular Weight; Mosaicism; Nature; Outcome; Parasites; Pharmacotherapy; Prevention; Protons; Reading; 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; 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和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序列的进展，包括克氏锥虫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）中极端的全基因组杂合性，以及参考菌株中的遗传嵌合现象。因此，当前克氏锥虫的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——每条染色体呈现为两个版本，在基因内容、两个染色体序列对齐的序列同一性、甚至成对的两个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针对这种人类疾病病原体的全基因组测序研究的功效和力量，并且几乎使任何涉及审查参考基因组的努力变得困难且常常徒劳无功。我们建议使用最初用于生成它的方法的三个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 参考基因组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克氏锥虫分离株进行测序，以简化组装。第三，由于所有克氏锥虫系均源自两个祖先、发散、纯合、非嵌合谱系，因此我们将为每个“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。