Deconvolution and Assembly of Metgenomes Using Chromatin Conformation Capture

 描述(申请人提供)：我们的身体、土壤和地球上已知的所有生态系统中微生物群落(“微生物区系”)的多样性直到最近才被实现。在多个身体部位发现了不同的微生物区系--其中一些提供健康益处，另一些则导致疾病。对这些群落的分析将揭示确定疾病易感性的新方法，并将使操纵人类微生物区系以优化人类健康成为可能。在植物细胞内和周围发现了其他微生物区系，其中一些提供营养和抗病益处，而另一些则会导致疾病。了解植物微生物区系将确定和定义新的生态兼容和可持续的农业实践，并使农业能够扩展到目前不适合的土地。此外，所有生态系统中前所未有的微生物多样性提供了巨大的、未知的遗传多样性，具有深远的工业应用。技术障碍：我们掌握的有关微生物区系的大部分信息来自高通量测序技术。因为绝大多数微生物是未知的，不能被提纯，所以微生物区系中的微生物必须保持混合，并进行共同测序(“元基因组学”)。使用常规方法，很难破译哪些序列属于特定微生物(“去卷积”)，因为有关起源细胞的信息在细胞破裂和DNA测序准备时丢失--特别是对于具有多条染色体或质粒的复杂基因组。我们的技术进步：我们使用“染色体构象捕捉”，在破碎细胞和处理DNA之前，使染色体内和染色体间的DNA交联(稳定连接)。这使我们能够知道在去卷积过程中哪些序列起源于同一细胞。在我们的初步研究中，我们成功地组装了人工混合的微生物种群(真菌、细菌和古菌物种)，包括那些具有复杂基因组的微生物。假设：我们假设我们的方法可以应用于未知浓度的未知物种的自然微生物区系。具体目标：1)使湿实验室染色体构象捕获方法适用于真实世界的元基因组样本，包括困难和低生物量样本；以及2)开发生产质量软件，优化用于组装低丰度基因组、菌株去卷积和质粒分配。总体影响：完成后，我们相信我们的方法将成为元基因组测序的标准--以更好地实现人类、植物和生态系统微生物区系发现所提供的潜力。

 DESCRIPTION (provided by applicant): The diversity of communities of microorganisms ("microbiota") in our bodies, in soils, and throughout all ecosystems known on Earth has only recently been realized. Different microbiota are found at multiple body sites - where some provide health benefits and others cause disease. Analysis of these communities will reveal new ways to determine predisposition to diseases and will enable manipulation of the human microbiota to optimize human health. Other microbiota are found in and around plant cells, where some provide nutritional and anti-disease benefits, while others cause disease. Understanding plant microbiota will identify and define new ecologically compatible and sustainable agricultural practices and enable agricultural expansion to currently unsuitable land. Furthermore, the unprecedented diversity of microorganisms throughout all ecosystems provides for tremendous and uncharted genetic diversity, with far-reaching industrial applications. Technology Hurdle: Much of the information we have about microbiota derives from high-throughput sequencing technologies. Because the vast majority of microorganisms are unknown and cannot be purified, the microorganisms in microbiota must remain mixed and are co-sequenced ("metagenomics"). Using conventional methods, it is difficult to decipher which sequences belong to specific microorganisms ("deconvolution") because information regarding the cell of origin is lost upon breaking cells and preparation of DNA for sequencing - especially for complex genomes with multiple chromosomes or plasmids. Our Technological Advance: We used "chromosome conformation capture", to make both intra- and inter-chromosomal DNA crosslinks (stable linkages) - prior to breaking cells and processing of DNA. This allowed us to know which sequences originated in the same cell during deconvolution. In our initial studies, we successfully assembled artificially mixed populations of microorganisms (fungal, bacterial, and archaeal species), including those with complex genomes. Hypothesis: We hypothesize that our method can be applied to natural microbiota with unknown species at unknown concentrations. Specific Aims: 1) To adapt wet-lab chromosome conformation capture methods to real-world metagenomic samples, including difficult and low-biomass samples; and 2) develop production-quality software optimized for assembling low-abundance genomes, strain deconvolution, and plasmid assignment. Overall Impact: Upon completion, we believe our methods will become the standard for metagenomic sequencing - to better realize the potential that discoveries of human, plant, and ecosystem microbiota have to offer.