High Accuracy Single Molecule DNA Sequencing by Synthesis

描述(申请人提供)：Helicos生物科学公司开发了一种全自动仪器，能够在平面上对单分子DNA进行测序。Helicos现在正在开发这种能力的高通量版本，用于对整个人类基因组进行重新测序。测序策略包括从数十亿股DNA中获得短读数(约25个碱基)，这些DNA链固定在试剂流细胞的表面上。该研究计划旨在解决这一策略的某些局限性，以高精度对高度可变的基因组进行重新测序，并最终重新组装从未测序过的基因组。高精度测序的能力将通过启用两遍测序策略(具体目标1)来实现。这需要从同一链上的相同位置获得两个读数。两次通过策略将需要以稳定和生物相容的方式将模板链以共价方式连接到表面。其次，将通过开发成对末端读取方法(具体目标2)来解决对高度可变或异常基因组进行测序的能力。这涉及到从同一条DNA链上获得两个约25个碱基的末端读数。两次读数之间的距离将被限制在一个范围内，这是由于限制了天然核苷酸的添加。从这一补充信息中获益所需的生物信息学将与样品准备和测序工作并行发展。最后，通过将两遍测序策略与配对末端读取策略(特定目标3)相结合，将实现从头开始基因组组装。这将导致产生实际上很长的读数，由来自表面相同DNA链的多个25碱基读数组成。前两个具体目标所取得的进展，将大大有助于第三个目标的成功。所有实验都将使用原型单分子测序仪进行。我们最后将通过测序和组装一个平均深度为10倍、覆盖率为90%、错误率为0.5%的4Mb细菌基因组从头开始测序和组装这些策略的实用性。这项研究计划的长期目标(在资助的10年内)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异常，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系统将实现目前技术过于昂贵或难以实现的应用，包括：对正常或肿瘤组织的整个人类基因组进行测序，对表观遗传学变化进行全基因组评估，以及对数千种正常和疾病组织类型的数字表达图谱。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.