Construction of A Reference Sequence Data Set For The Human Microbiome Project

人类的生理和健康高度依赖于生活在人体内和人体内多个生态环境中的数十亿种微生物的活动。这些微生物很少有特征，很大程度上是因为大多数微生物还没有培养出来。因此，这大量物种对人类健康的影响仍有待阐明。这项建议介绍了J.Craig Venter研究所生成大量与人体相关的参考物种的基因组序列的方法，这些参考物种包括细菌、真菌、病毒和噬菌体。我们还描述了如何从从多个个体的不同身体部位收集的DNA样本中生成元基因组序列。 具体目标包括400个微生物基因组的测序、组装和注释(其中60个将关闭)，以提供元基因组数据的参考基因组；通过对这些样本进行鸟枪式测序来表征来自多个身体部位的微生物区系，以及使用我们最先进的组装和分析工具来进一步解释这些数据。我们还将研究微真核生物的多样性，重点是28S rRNA基因和内部转录间隔区。细胞分选，结合全基因组扩增，将被用来从以前未培养的原核生物中获得基因组DNA。最后，通过元基因组分析确定的标记基因表达的RNA的动态范围将使用一种新的mRNA转录捕获和扩增方法进行询问。 这项研究将使用尖端技术进行，包括下一代测序方法。我们将继续对研究临床医生进行推广和教育，并将使所有数据随时可用。毫无疑问，这项拟议研究的结果将增加目前对与人体相关的微生物多样性以及这些物种对健康和疾病的影响的了解。

Human physiology and health is highly dependent on the activities of billions of microorganisms that inhabit multiple niches within and on the human body. Few of these microorganisms have been characterized, in large part because the majority are yet to be cultivated. As such, the impact this vast number of species has on human health remains to be elucidated. This proposal presents the J. Craig Venter Institute's approach to generating the genomic sequences of a significant number of reference species including bacteria, fungi, viruses and phage that are associated with the human body. We also describe how we will generate metagenomic sequences from DNA samples collected from various body sites of multiple individuals. The specific aims include the sequencing, assembly and annotation of 400 microbial genomes (60 of which will be closed) to provide reference genomes for metagenomic data; characterization of the microbiota from multiple body sites via shotgun sequencing of these samples, and the use of our state-of-the-art assembly and analysis tools to further interpret this data. We will also investigate microeukaryotic diversity, focusing on the 28S rRNA gene and internal transcribed spacer regions. Cell sorting, combined with whole genome amplification will be employed to obtain genomic DNA from previously uncultured prokaryotes. Finally, the dynamic range of expressed RNA from marker genes identified through metagenomic analyses will be interrogated using a novel mRNA transcript capture and amplification approach. This study will be conducted using cutting edge technologies including next generation approaches to sequencing. We will continue outreach and education to research clinicians, and will make all data readily available. Without a doubt, the findings from the proposed study will increase current understanding of the microbial diversity associated with the human body and the impact of these species on health and disease.