Phased Sequencing of Complete Genomes via DNA Barcode Array

通过 DNA 条形码阵列对完整基因组进行定相测序

• 批准号: 8906555
• 负责人: Filip Crnogorac
• 金额: $ 35万
• 依托单位: CENTRILLION BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-07 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8906555
• 关键词: Address; Binding; Bioinformatics; Biological Sciences; Chromosomes; Code; Comb animal structure; Computer software; DNA; DNA Microarray Chip; DNA Sequence; Detection; Diploidy; Genome; Genomics; Glass; Goals; Haplotypes; Human Genome; Immobilization; Individual; Length; Libraries; Location; Methods; Nucleotides; Oligonucleotides; Phase; Polyploidy; Positioning Attribute; Process; Reading; Reagent; Relative (related person); Research; Resolution; Sampling; Sequence Analysis; Small Business Innovation Research Grant; Stretching; Structure; Surface; Surface Properties; Technology; Variant; base; cost; experience; genetic variant; genome sequencing; human genome sequencing; innovation; innovative technologies; insertion/deletion mutation; instrumentation; nanopore; next generation sequencing; personalized medicine; public health relevance; research and development; scaffold

 DESCRIPTION (provided by applicant): The goal of this SBIR application is to develop innovative technologies to provide more complete whole genome sequences, which accurately identify all genetic variants (single nucleotide, insertions/deletions, polyploidy, structural variants) and phase the variants to the appropriate homologous chromosome. The approach for solving these long standing challenges in genomics is tremendously significant and will transform the way genomes are sequenced and analyzed. The basic approach is to stretch many individual DNA molecules on an oligonucleotide chip. These DNA molecules will be primed by the oligonucleotides on the chip, which will serve as templates for traditional Next Generation Sequencing (NGS). The oligonucleotide on the chip is barcoded to identify the location on the chip, and thus provide a scaffold for assembling the short NGS reads (i.e. from an Illumina HiSeq). Scaffolding the short reads will solve key problems in genomics by allowing for high quality de novo assembly with very accurate single nucleotide variant detection, structure variant detection, and resolution of haplotypes from diploid samples. The aims of the proposal are to address the critical challenges in this process. First, in Aim 1 we will optimize the DNA chip fabrication to (a) reduce the feature size and pitch, (b) reverse the orientation of the oligos to make 3' end free for extension, and (c) increase the length and accuracy of the oligosynthesis. Secondly, in Aim 2 we will develop the approach for combing chromosomal DNA on oligonucleotide chip surfaces. We have experience combing DNA on traditional glass surfaces; however, the different surface properties of the DNA chip will likely pose new challenges for the DNA combing. Finally, in Aim 3 we will generate sequencing libraries from the immobilized DNA and sequence the libraries on an Illumina HiSeq. We will also sequence the barcodes on the Illumina platform, which will provide a scaffold for assembling the short reads. In summary, this project will integrate several highly innovative and breakthrough technologies to address major limitations of current next generation sequencing.

 描述（由申请人提供）：本SBIR申请的目标是开发创新技术，以提供更完整的全基因组序列，准确识别所有遗传变异（单核苷酸、插入/缺失、多倍性、结构变异），并将变异定相至适当的同源染色体。解决基因组学中这些长期存在的挑战的方法是非常重要的，并将改变基因组测序和分析的方式。 基本方法是在寡核苷酸芯片上拉伸许多单个DNA分子。这些DNA分子将由芯片上的寡核苷酸引发，这些寡核苷酸将作为传统下一代测序（NGS）的模板。将芯片上的寡核苷酸条形码化以鉴定芯片上的位置，并因此提供用于组装短NGS读数（即来自Illumina HiSeq）的支架。支架化短读段将通过允许具有非常准确的单核苷酸变体检测、结构变体检测和来自二倍体样品的单倍型解析的高质量从头组装来解决基因组学中的关键问题。 该提案的目的是应对这一进程中的关键挑战。首先，在目标1中，我们将优化DNA芯片制造以（a）减小特征尺寸和间距，（B）反转寡核苷酸的取向以使3'端自由延伸，以及（c）增加寡核苷酸合成的长度和准确性。其次，在目标2中，我们将开发在寡核苷酸芯片表面上结合染色体DNA的方法。我们有在传统玻璃表面梳理DNA的经验;然而，DNA芯片的不同表面性质可能会对DNA梳理提出新的挑战。最后，在目标3中，我们将从固定的DNA生成测序文库，并在Illumina HiSeq上对文库进行测序。我们还将在Illumina平台上对条形码进行测序，这将为组装短读段提供支架。 总之，该项目将整合几项高度创新和突破性的技术，以解决当前下一代测序的主要局限性。