High Accuracy Single Molecule DNA Sequencing by Synthesis

描述（由申请人提供）：Helicos生物科学公司开发了一种全自动仪器，能够对平面上的DNA单分子进行测序。Helicos目前正在开发一种高通量版本，用于对整个人类基因组进行重新测序。测序策略包括从固定在试剂流动池内表面的数十亿条DNA链中获得短读数（约25个碱基）。该研究计划旨在解决该策略的某些局限性，以便以高精度重新测序高度可变的基因组，并最终重新组装从未测序过的基因组。通过启用双通道测序策略（Specific Aim 1），可以实现高精度测序。这需要从同一条链上的相同位置获得两次读取。双通道策略将需要模板链以稳定和生物相容性的方式共价附着在表面。其次，对高度可变或异常基因组进行测序的能力将随着成对末端读取方法的发展而得到解决（Specific Aim 2）。这包括从同一条DNA链上获得两个远端约25个碱基的读取。由于天然核苷酸的有限加入，两个reads之间的距离将被限制在一定范围内。从这些补充信息中获益所需的生物信息学将与样品制备和测序工作并行开发。最后，通过将双通道测序策略与成对端读策略相结合，可以实现从头基因组组装（Specific Aim 3）。这将导致产生实际上很长的reads，由多个25个碱基的reads组成，这些reads来自于表面上的同一条DNA链。在前两个具体目标方面取得的进展将大大有助于第三个目标的成功。所有实验将使用原型单分子测序仪器进行。最后，我们将通过重新测序和组装一个平均深度为10X，覆盖率为bbb90 %，错误率<0.5%的4mb细菌基因组来证明这些策略的实用性。这项研究计划的长期目标（在10年的资金支持下）是开发一个强大的单分子测序系统，该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，满载成本为1000美元，每台仪器每天可处理8个基因组。因此，Helicos系统将使当前技术无法实现的昂贵或困难的应用成为可能，包括：从正常或肿瘤组织中提取整个人类基因组的测序，全基因组表观遗传变化的评估，以及数千种正常和病变组织类型的数字表达谱。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，并将导致在疾病的诊断、治疗和预防中使用基因组信息。

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.