SEQUENCING THE MICROBIAL COMMUNITY AT THE WESTERN CHANNEL OBSERVATORY / SEASONAL CHANGES IN DIVERSITY AND FUNCTION

海洋生态系统对地球系统的运行至关重要。它在改变气候方面发挥着至关重要的作用，它们占地球年初级生产量的一半，我们星球上约三分之二的商品和服务是由沿海和开阔海洋生态系统提供的。这些环境由细菌和古生菌等微生物主导，它们驱动着全球所有的生物地球化学循环，并通过产生具有化学活性的生物气酶对大气产生直接影响。因此，海洋的生物地球化学是微生物活动的结果，了解地球系统需要对海洋微生物进行大量研究。大量的微生物及其巨大的新陈代谢多样性表明，遗传多样性和功能远远高于迄今所研究的。克雷格·文特尔(Venter等人，2004年)的研究首次证明了海洋环境中微生物多样性的巨大范围，他使用Sanger序列分析了从数百升马尾藻海水中产生的枪炮克隆库。这项极其昂贵的序列分析在1800种微生物中发现了120万个新基因，其中包括150种新细菌。他们在马尾藻中发现的基因数量之多令人震惊，并进一步支持了海洋中微生物生命远比预期更丰富和多样化的观点。为了衡量这种多样性水平，有必要将其与人类基因组/目前被认为由20,000-25,000个基因组成的基因的数量进行比较。这项建议是为了扩大PGP资助的水生微生物元基因组学项目的成果，因为它利用了最近可用的焦磷酸测序的新测序方法，该方法由454生命科学公司(美国)开发。典型的焦磷酸测序从一个样本中产生大约400,000个序列。由于这是目前获得资金的PGP项目的延伸，我们不要求支付工资费用。我们要求指导委员会考虑提供100%的排序成本(目前NERC规则只允许80%)。我们建议使用新的高通量焦磷酸测序方法来分析NERC资助的西海峡天文台(WCO)细菌和古菌的遗传多样性和功能。我们将使用这种方法来评估以季节性周期为特征的组成和功能的变化。建议的焦磷酸测序方法的输出将产生约320万个序列读数。基于250个碱基的平均阅读长度，这将产生大约8亿个碱基的遗传信息，包括大约200-400个细菌基因组等价物。总而言之，重要的是要理解这个项目在两个方面是新颖的。首先，该项目将首次大规模分析同一样本中微生物的多样性和活性，并评估不同季节对两者的影响。其次，我们将采用的分子方法首次使用新的高通量焦磷酸测序技术来分析整个微生物群落的基因表达。

The marine ecosystem is crucial for the functioning of the Earth system. It plays an essential part in modifying the climate, they account for half of the annual primary production on the planet and about two thirds of goods and services from our planet are provided by coastal and open ocean ecosystems. These environments are dominated by microorganisms, such as bacteria and archaea, which drive all global biogeochemical cycles and have a direct influence on the atmosphere by the production of chemically-active biogases. Therefore the biogeochemistry of the oceans is the consequence of microbial activity and understanding the Earth System requires a significant research effort on marine microbes. The enormous number of microbes and their vast metabolic diversity, suggests that the genetic diversity and function is far higher than that investigated so far. The huge extent of the microbial diversity in the marine environment was first demonstrated by the study of Craig Venter (Venter et al., 2004) who used Sanger sequence analysis of a shot-gun clone library produced from of a few hundred litres of the Sargasso Sea water. This extremely expensive sequence analysis found 1.2 million new genes within 1800 species of microbes, including 150 new species of bacteria. The sheer number of genes they found in the Sargasso is astounding and further supports the notion that the microbial life in the ocean is far more abundant and diverse than expected. In order to scale this level of diversity it is relevant to compare it with the number of genes in the human genome / currently thought to comprise 20,000 - 25,000 genes. This proposal is to extent the output from the PGP-funded project on Aquatic Microbial Metagenomics in that it utilises on the recently available novel sequencing methodology of pyrosequencing, which has been developed by 454 Life Sciences, Inc. (USA). A typical pyrosequencing run generates ca. 400,000 sequences from one sample. As it is an extention of the currently funded PGP project we do not request salary costs. We request the steering committee to consider providing 100% of the sequencing costs (currently NERC rules only allow 80%). We propose to use the novel high-throughput pyrosequencing approach to analyse both the genetic diversity and function of the bacteria and archaea at the NERC-funded Western Channel Observatory (WCO). We will use this approach to assess the changes in the composition and function that are characterised by seasonal cycles. The output of the proposed pyrosequencing approach will generate some 3.2 million sequence reads. Based on an average read length of 250 bp, this will produce approximately 800 million bp of genetic information comprising approximately 200 - 400 bacterial genome equivalents. In conclusion, it is important to understand that this project is novel in two points. Firstly, and for the first time on a large scale this project will analyse both the diversity and the activity of the microbes present in the same sample, as well as assessing the influence of different seasons on both. And secondly, the molecular approach that we will employ uses for the first time the new high-throughput pyrosequencing technique to analyse the gene expression of a whole microbial community.