Thermostable primer-free whole genome amplification of single cells with viral en

描述（由申请人提供）：疾病的遗传基础仍然是一个重要的研究问题，影响癌症生物学，神经学，心脏病学，发育，甚至微生物学领域，最近由人类微生物组计划实现。确定所观察到的表型背后的基因组序列现在很普遍。基因组测序变得更快，更便宜，允许获得更多的基因组信息和更好地了解基因型和表型之间的关系。这些技术正越来越多地应用于越来越小的样本大小，甚至单个细胞。单个细胞包含所有必要的遗传信息，理论上可以重复扩增。然而，从这样小的样品产生足够的扩增DNA用于基因组测序受到当前技术的几个特征的限制。目前的扩增方案使用phi29聚合酶和用随机六聚体引发的DNA。虽然该系统允许使用单细胞研究疾病，但它有许多缺点。其中主要的是引物扩增假象和高偏倚，当靶DNA的量低时，高偏倚尤其加剧。单基因组的偏倚扩增导致序列信息丢失，如等位基因缺失，并使拷贝数变异分析复杂化。这种偏差是随机的，因为它是随机六聚体初始退火从随机区域开始扩增的结果。这种随机偏差使得单细胞之间的比较几乎不可能。该第一阶段提案的目标是实现无引物DNA合成，以大大提高从小至单细胞的样品扩增DNA的效率和覆盖率。去除引物将消除非生产性副反应并减少最终产物中的偏差。预期该系统在较高温度下等温操作，而不需要初始变性步骤。我们提出的研究的商业化结果将是一种用于高保真、低偏倚（高覆盖率）扩增单细胞DNA的试剂盒，而没有引物背景问题。这项工作将通过提供更高的序列覆盖率来推进单细胞基因组学的研究，从而能够研究癌症，干细胞，生物膜和人类微生物组。

DESCRIPTION (provided by applicant): The genetic basis of disease remains a significant research problem, impacting the fields of cancer biology, neurology, cardiology, development, and even microbiology, as recently realized by the Human Microbiome Project. Determination of the genomic sequence underlying observed phenotypes is now widespread. Genomic sequencing is becoming faster and cheaper, allowing access to more genomic information and greater understanding of the relationships between genotype and phenotype. Increasingly, these technologies are being applied to smaller and smaller sample sizes, down to single cells. A single cell contains all the necessary genetic information, and theoretically can be amplified repeatedly. However, generating enough amplified DNA from such small samples for genomic sequencing is constrained by several features of current technologies. Current amplification protocols use phi29 polymerase and DNA primed with random hexamers. Although this system has allowed for the study of disease using single cells, it has numerous disadvantages. Primary among these are primer amplification artifacts and high bias which is especially exacerbated when the amount of target DNA is low. Biased amplification from single genomes results in loss of sequence information such as allelic dropout and complicates copy number variant analysis. This bias is stochastic because it results from the initial annealing of random hexamers to begin amplification from random regions. Such stochastic bias makes comparison between single cells nearly impossible. The goal of this Phase I proposal is to enable primer-free DNA synthesis, to vastly improve the efficiency and coverage of amplified DNA from samples as small as single cells. Removing primers will eliminate unproductive side-reactions and decrease bias in the final product. This system is expected to operate isothermally at higher temperatures, without the need for an initial denaturation step. The commercial result of our proposed study will be a reagent kit for high fidelity, low bias (high coverage) amplification of DNA from single cells without primer background issues. This work will advance the study of single cell genomics by providing higher sequence coverage, enabling research into cancer, stem cells, biofilms, and the human microbiome.