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 开发了一种全自动仪器，能够对平面上的单分子 DNA 进行测序。 Helicos 目前正在开发这种功能的高通量版本，用于对整个人类基因组进行重新测序。测序策略涉及从固定在试剂流通池内表面上的数十亿条 DNA 链中获取短读数（约 25 个碱基）。该研究计划旨在解决该策略的某些局限性，以高精度对高度可变的基因组进行重新测序，并最终从头组装以前从未测序过的基因组。通过启用两遍测序策略（具体目标 1），可以实现高精度测序的能力。这需要从同一条链上的同一位置获得两个读数。两遍策略需要将模板链以稳定且生物相容的方式共价连接到表面。其次，对高度可变或异常基因组进行测序的能力将通过双端读取方法的开发得到解决（具体目标 2）。这涉及从同一条 DNA 链获取两个远端约 25 个碱基的读数。通过限制添加天然核苷酸，两个读数之间的距离将被限制在一定范围内。从这些补充信息中受益所需的生物信息学将与样品制备和测序工作同时开发。最后，通过将两遍测序策略与双端读取策略相结合，可以实现从头基因组组装（具体目标 3）。这将导致生成实际上很长的读数，由表面同一条 DNA 链的多个 25 个碱基读数组成。前两个具体目标取得的进展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。我们最终将通过从头测序和组装 4 Mb 细菌基因组，平均深度为 10X，覆盖率 >90% 和错误率 <0.5% 来证明这些策略的实用性。该研究计划的长期目标（资助后 10 年内）是开发一个强大的单分子测序系统，能够对人类基因组进行测序，并检测该基因组中的所有遗传变异和畸变，以实现满载的目标。成本为 1000 美元，每台仪器每天可处理 8 个基因组。因此，Helicos 系统将能够实现那些成本太高或难以用现有技术实现的应用，包括：对正常或肿瘤组织的整个人类基因组进行测序、表观遗传变化的全基因组评估以及数千个基因的数字表达谱分析。正常和患病的组织类型。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展，并将导致基因组信息在疾病的诊断、治疗和预防中的使用。