High Accuracy Single Molecule DNA Sequencing by Synthesis

高精度单分子 DNA 合成测序

• 批准号: 7192686
• 负责人: TIMOTHY D HARRIS
• 金额: $ 66.38万
• 依托单位: HELICOS BIOSCIENCE CORPORATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-28 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192686
• 关键词: genome; nucleic acid sequence; reading

DESCRIPTION (provided by applicant): Helicos BioSciences Corporation has developed a fully automated instrument capable of sequencing single molecules of DNA on a planar surface. Helicos is now developing a high-throughput version of this capability for the re-sequencing of whole human genomes. The sequencing strategy involves obtaining short reads (about 25 bases) from billions of strands of DNA, immobilized on a surface inside a reagent flow cell. The research plan aims to address certain limitations of this strategy for the re-sequencing of highly variable genomes with high accuracy, and for the eventual de novo assembly of never before sequenced genomes. The ability to sequence with a high-accuracy will be achieved by enabling a two-pass sequencing strategy (Specific Aim 1). This entails obtaining two reads from the same position on the same strand. A two-pass strategy will require covalent attachment of template strands to the surface in a stable and biocompatible fashion. Secondly, the ability to sequence highly variable or aberrant genomes will be addressed with the development of a paired-end read approach (Specific Aim 2). This involves acquiring two distal about 25 base reads from the same strand of DNA. The distance between the two reads will be limited to a range by the restricted addition of natural nucleotides. The bioinformatics required to benefit from this supplemental information will be developed in parallel with the sample preparation and sequencing efforts. Lastly, de novo genome assembly will be enabled by combining the two-pass sequencing strategy to the paired-end read strategy (Specific Aim 3). This will result in the generation of virtually long reads, composed of multiple 25 base reads from the same strand of DNA on the surface. The advances made in the two previous specific aims, will contribute greatly to the success of this third aim. All experiments will be carried out using the prototype single molecule sequencing instruments. We will finally demonstrate the utility of these strategies by sequencing and assembling a 4 Mb bacterial genome de novo with an average depth of 10X, a coverage of >90% and an error rate of <0.5%. The long-term objective of this research plan (within 10 years of funding) is to develop a robust single molecule sequencing system, which is capable of sequencing a human genome, and detecting all genetic variations and aberrations in that genome, for a fully loaded cost of $1000, at a throughput of 8 genomes per instrument per day. The Helicos system will thereby enable applications that are too costly or difficult to carry out with current technologies including: the sequencing of whole human genomes from normal or tumor tissues, the genome-wide assessment of epigenetic changes, and the digital expression profiling of thousands of normal and diseased tissue types. Ultimately these methods will yield advances in the fields of cancer and complex disease genetics/genomics, and will result in the use of genomic information in the diagnosis, treatment and prevention of disease.

描述（由申请人提供）：Helicos Biosciences Corporation已开发了一种能够在平面表面上测序DNA的单分子的全自动仪器。 Helicos现在正在开发此能力的高通量版本，以重新续订整个人类基因组。测序策略涉及从数十亿链DNA中获得简短的读数（约25个碱基），并固定在试剂流动池内的表面上。该研究计划旨在解决此策略的某些局限性，以高准确性重新测序高度可变的基因组，以及最终从未测序的基因组的从头组装。通过启用两通序列策略，将实现具有高准确率的测序能力（特定目标1）。这需要从同一链上的同一位置获得两个读取。两次通行的策略将需要以稳定且具有生物相容性的方式将模板链共价附着在表面上。其次，将通过配对读取方法的开发来解决高度可变或异常基因组的测序能力（特定目标2）。这涉及从相同的DNA链中获取大约25个底座的两个远端读数。通过限制自然核苷酸的限制，两个读数之间的距离将限制为范围。受益于此补充信息所需的生物信息学将与样本准备和测序工作并行开发。最后，通过将两次通行测序策略结合到配对末端读取策略（特定的AIM 3），将启用从头基因组组装。这将导致几乎长期读取的产生，由多个25个基本读数组成，从表面上的同一DNA链读取。前两个具体目标中取得的进步将为第三个目标的成功做出巨大贡献。所有实验将使用原型单分子测序仪器进行。我们最终将通过测序和组装4 MB细菌基因组DE的效用，平均深度为10倍，覆盖率> 90％，错误率为<0.5％。该研究计划的长期目标（资金的10年之内）是开发一个可靠的单分子测序系统，该系统能够对人类基因组进行测序，并以每天8个基因组的吞吐量为1000美元。因此，Helicos系统将实现对当前技术的成本太高或难以执行的应用，包括：从正常或肿瘤组织中对整个人类基因组进行测序，对表观遗传变化的全基因组的评估以及数千种正常和患病的组织类型的数字表达分析。最终，这些方法将在癌症和复杂疾病遗传学/基因组学领域取得进步，并将导致在诊断，治疗和预防疾病的诊断，治疗和预防中使用基因组信息。