Dynamics of DNA Barcodes in Nanochannels

 描述：基因组变异性出现在一系列尺度上，从单个核苷酸到整个染色体。结构变异，如易位和倒位，通常涉及数十个DNA酶对重排为DNA的兆碱基对。虽然下一代测序已经彻底改变了基因组学的许多领域，但由于读取长度短，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，迫切需要提供大规模基因组信息（数百万个碱基）的基因组作图技术，以补充在Illumina短读测序平台上生成的金标准（数百个碱基）。在第一个三年期间， R01奖，明尼苏达大学和BioNano Genomics一直在合作建立这样一种技术的基本基础：纳米通道阵列中的基因组图谱。纳米通道阵列中的基因组映射与大量完整的基因组DNA分子一起工作，这些分子的大小几乎达到百万碱基，已经用序列特异性标签进行了“条形码化”。这些条形码化分子通过限制在45 nm纳米通道中而延伸，并且通过荧光显微镜读取条形码。到目前为止，我们已经对标记DNA的热波动有了全面的了解，这设置了测量误差的下限，以及一套工具，使我们能够检测（和预测）纳米通道中DNA的物理重排。该奖项的下一个三年期建立在我们的进步，以实现两个新的具体目标。具体目标1超越DNA的均聚物模型，将序列依赖性微观力学纳入工程模型，用于预测器件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，其（i）将结构变异与GC含量相关联而不需要基因组序列，以及（ii）更好地解析其中切口条形码相似的基因组区域。具体目标2将通过调整缓冲液成分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基因组作图技术的创新工程，在此期间，我们利用两个团队的独特能力来推进我们对受限聚合物的基本理解，同时为新兴的基因组作图技术提供工程基础，并开发基因组作图的新功能。除了发表基本成果外，该项目还将通过将基因组作图技术的任何进展纳入BioNano Genomics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.