Development of a kit for the targeted depletion of abundant sequencesfrom DNA/RNA libraries

 描述(由申请人提供)：人类元基因组学--通过下一代测序研究我们身体的微生物和病毒群落--正在改变目前健康和疾病的范式。微生物群体的直接测序在传染病的诊断和流行病学以及加深我们对从肥胖到自身免疫性疾病等慢性常见病的病因、治疗和预防的了解方面具有巨大的潜力。出于这个原因，NIH在研究项目(如人类微生物组项目)上投入了大量资金，以确定一系列组织中健康和疾病个体的微生物组特征。然而，将这项工作转化为临床需要克服一个关键障碍：许多临床样本中存在低水平的微生物序列和高水平的人类序列，这降低了元基因组学方法的敏感性，并显著增加了成本。识别基因组学有一种新颖和创新的方法来定向耗尽测序文库中的人类序列。我们的第一阶段假设是，CRISPR-CAS9系统可以用来系统地和强有力地从测序文库中耗尽人类序列，以提高元基因组分析的灵敏度和成本效益。具体目标1是开发针对线粒体DNA和核糖体RNA的指导RNA面板，并使用这些模板来改进反应条件和指导RNA设计原则。具体目标2是开发一个转录组范围的指导RNA小组，以耗尽RNAseq文库，并根据临床样本进行验证。预期的结果是CRISPR-Cas9方法被证明可以从测序文库中耗尽80%的靶向人类序列，而非人类序列在一小时内丢失不到5%。第二阶段的工作将包括将耗尽效率提高到100%，并将该方法扩展到从DNA衍生测序文库中耗尽整个人类基因组。我们的技术直接集成到现有的工作流程中，我们的初步数据显示，我们可以在不到一小时的时间内将atac-seq文库中的mtDNA耗尽60%。将这项工作扩展到rRNA，然后是RNAseq，将为研究和临床社区产生一套工具，将加速在基于测序的临床诊断中采用元基因组测序。我们通过提交这项技术的专利申请来保护我们的知识产权，并找到了领先的学术合作伙伴，帮助我们在一系列样本上测试和验证我们的方法。最后，我们的执行领导层和董事会在社区中处于有利地位，可以通过现有的试剂盒制造商(包括新英格兰Biolabs)和领先的测序供应商(包括Illumina)帮助我们将这项技术商业化。

 DESCRIPTION (provided by applicant): Human metagenomics - the study of our body's microbial and viral communities through next generation sequencing - is transforming current paradigms of both health and disease. Direct sequencing of microbiome populations has enormous potential for direct use in the diagnosis and epidemiology of infectious disease and for furthering our understanding of the causes, treatment, and prevention of chronic common diseases, from obesity to autoimmune disorders. For this reason, the NIH has invested heavily in research projects (such as the Human Microbiome project) to characterize the microbiome of healthy and diseased individuals across a spectrum of tissues. Translating this work into the clinic, however, requires overcoming a key hurdle: low levels of microbial sequence and high levels of human sequence present in many clinical samples reduce the sensitivity and dramatically increase the cost of metagenomics approaches. IdentifyGenomics has a novel and innovative method for the targeted depletion of human sequences from sequencing libraries. Our Phase 1 hypothesis is that a CRISPR-Cas9 system can be used to systematically and robustly deplete human sequences from sequencing libraries to improve the sensitivity and cost-effectiveness of metagenomics analysis. Specific Aim 1 is to develop guide RNA panels targeted to mitochondrial DNA and ribosomal RNA and to use these to refine reaction conditions and guide RNA design principles. Specific Aim 2 is to develop a transcriptome-wide guide RNA panel to deplete RNAseq libraries and validate it against clinical samples. The expected outcome is a CRISPR-Cas9 method demonstrated to deplete 80% of targeted human sequences from a sequencing library, with less than 5% loss of non-human sequences in one hour. Phase II work will include improving depletion efficiencies towards 100% and extending the method to depletion of the whole human genome from DNA-derived sequencing libraries. Our technology integrates directly into existing workflows and our preliminary data shows we can deplete mtDNA from ATAC-seq libraries by 60% in under an hour. Extending this work to rRNA and then RNAseq will produce a suite of tools for the research and clinical community that will accelerate the adoption of metagenomics sequencing in sequencing-based clinical diagnostics. We have secured our intellectual property by filing patent applications on this technology and have identified leading academic partners to help us test and validate our methods on a range of samples. Finally, our executive leadership and board are well positioned in the community to help us commercialize this technology through existing kit manufacturers (including New England Biolabs) and leading sequencing providers (including Illumina).