Dynamics of DNA Barcodes in Nanochannels

 描述：基因组变异性出现在一系列尺度上，从单核苷酸到整个染色体。结构变异，如易位和倒位，通常涉及数万个碱基对的DNA重新排列为兆碱基对。虽然下一代测序已经给基因组学的许多领域带来了革命性的变化，但由于读取长度较短，它对结构变异的分析影响较小。由于癌症基因组的结构复杂性，当测序用于癌症诊断时，也会出现类似的问题。因此，迫切需要能够提供大规模基因组信息(数百万个碱基)的基因组图谱技术，以补充在Illumina短读测序平台(数百个碱基)上产生的黄金标准。在这一年的前三年 R01奖，明尼苏达大学和生物纳米基因组学一直在合作，为一项这样的技术建立基本基础：纳米通道阵列中的基因组图谱。纳米通道阵列中的基因组图谱与大量完整的基因组DNA分子一起工作，这些DNA分子的大小几乎是一兆个碱基，这些分子已经用序列特定的标签进行了“条形码”编码。这些条形码分子通过限制在45 nm纳米通道中延伸，并通过荧光显微镜读取条形码。到目前为止，我们已经对标记的DNA的热涨落有了全面的了解，这为测量误差设定了下限，以及一套允许我们检测(和预测)DNA在纳米通道中的物理重排的工具。该奖项的下一个三年期建立在我们取得进展的基础上，以实现两个新的具体目标。具体目标1超越了DNA的均聚物模型，将依赖于序列的微观力学结合到工程模型中，用于预测设备性能和分析实验数据。相应的实验开辟了一种新的测量模式，它(I)在不需要基因组序列的情况下将结构变化与GC含量联系起来，(Ii)更好地解析基因组中刻痕条形码相似的区域。具体目标2将通过调整缓冲器组成来优化测量分辨率和吞吐量之间的平衡，从而提高设备性能。在完成这些SA的过程中，我们将从第一个资助期开始继续进行基因组图谱技术的创新工程，在此期间，我们利用两个团队的独特能力来促进我们对受限聚合物的基本理解，同时为新兴的基因组图谱技术和开发基因组图谱的新功能提供工程基础。除了公布基本结果外，这个项目还将通过将基因组图谱技术的任何进步纳入BioNano Genology的下一代产品以及公开发布该项目产生的DNA模拟和数据分析软件来影响整个社区。

 DESCRIPTION: Genomic variability arises at a range of scales, from single nucleotides to entire chromosomes. Structural variations, such as translocations and inversions, typically involve rearrangements of tens of kilobase pairs to megabase pairs of DNA. While next-generation sequencing has revolutionized many areas of genomics, it has had less impact on the analysis of structural variations due to short read lengths. Similar issues arise when sequencing is used for cancer diagnosis, owing to the structural complexity of cancer genomes. Thus, a pressing need exists for genome mapping technologies that provide large-scale genomic information (millions of bases) that complements the gold standard generated on the Illumina short-read sequencing platform (hundreds of bases). In the first three-year period of this R01 award, the University of Minnesota and BioNano Genomics have been collaborating to establish the fundamental basis for one such technology: genome mapping in nanochannels arrays. Genome mapping in nanochannel arrays works with massive intact genomic DNA molecules, up to almost a megabase in size, that have been "barcoded" with sequence-specific labels. These barcoded molecules are extended by confinement in a 45 nm nanochannel, and the barcode is read by fluorescence microscopy. To date, we have developed a comprehensive understanding of the thermal fluctuations of the labeled DNA, which set the lower bound on the measurement error, as well as a suite of tools that allow us to detect (and predict) physical rearrangement of DNA in the nanochannel. The next three- year period of this award builds on our advances to accomplish two new Specific Aims. Specific Aim 1 moves beyond homopolymer models of DNA to incorporate sequence-dependent micromechanics into the engineering models for both prediction of device performance and analysis of experimental data. The corresponding experiments open up a new measurement modality that (i) associates structural variations with GC content without requiring the genome sequence and (ii) better resolves regions of the genome where the nicking barcodes are similar. Specific Aim 2 will lead to improved device performance by tuning the buffer composition to optimize the balance between measurement resolution and throughput. In completing these SAs, we will continue the innovative engineering of genome mapping technologies from the first grant period, where we leverage the unique capabilities of both teams to advance our fundamental understanding of confined polymers while providing an engineering basis for the emerging genome mapping technology and developing new functionalities for genome mapping. In addition to publishing fundamental results, this project will impact the community at large through incorporation of any advances in genome mapping technology in the next-generation products from BioNano Genomics and the public release of DNA simulation and data analysis software arising from the project.