Illumina Sequencer to Facilitate Functional Genomics at Berkeley

描述(由申请人提供)： 6.项目摘要/摘要。第二代DNA测序机能够以适度的成本在一次运行中产生数千万到数亿个序列读数，已经改变了功能基因组学。现在，直接通过测序分析复杂核酸混合物的组成比通过与DNA微阵列杂交间接分析复杂的核酸混合物更简单、更准确，而且在许多情况下成本更低，这是过去十年的标准。高通量测序也使新的功能基因组实验成为可能，而这些实验无法用微阵列有效地进行。加州大学伯克利分校的文森特·J·科茨基因组测序实验室通过为伯克利大学的教职员工提供下一代测序，以及在实验设计、样品制备和数据分析方面的培训和协助，促进了这一转变。该中心的两台Illumina基因组分析仪自上线以来一直在使用(一台在15个月前，另一台在9个月前)。在此期间，美国国立卫生研究院资助的23个(总共27个)实验室使用这些机器进行了各种功能基因组实验，包括：量化信使核糖核酸丰度、测量信使核糖核酸合成和衰减率、确定不同剪接形式及其使用频率、比较不同等位基因的表达、绘制转录因子和其他DNA相互作用蛋白结合区的位置、确定表观遗传标记在全基因组范围内的分布、编目RNA结合蛋白结合的RNA、精确定位突变体和从大的杂交中对个体进行基因分型。分析样本来自多种细菌、原生动物、植物、真菌和动物，包括模型和非模型系统，小鼠和人类干细胞，以及正常和患病的人类细胞系和组织。此外，伯克利分校的研究人员开发了新的基于测序的技术来表征DNA结合蛋白与其靶标的体外亲和力，并合理地进化出具有所需功能的蛋白质。该中心已被用于对几种细菌、真菌和动物基因组进行测序。支持这一系列活动的是伯克利的一群才华横溢的计算生物学家，他们正在开发创新的方法，用于碱基调用、阅读映射、组装和分析这种高通量测序数据，并分发和支持他们的方法的软件实现，这些方法在伯克利和其他地方得到了积极的应用。有了相当稳定的实验方案和分析方法，限制这项技术在伯克利广泛应用的主要因素是测序能力。我们的两台机器连续运行，平均每台机器每月产生10次运行(运行的长度取决于所需的读取长度和配对结束状态)。尽管如此，样品的队伍越来越长，目前处理样品的时间延误了近两个月。我们正在申请资金，以购买额外的Illumina基因组分析仪IIx DNA测序仪，以满足伯克利生物界已证明的需求。我们拥有一家全面运营的工厂，拥有一位杰出的总监，一年多来一直从Illumina测序仪生产高质量的数据。一台新机器将立即投入使用，并将对美国国立卫生研究院资助的社区研究产生重大积极影响。

DESCRIPTION (provided by applicant): 6. Project Summary/Abstract. Second generation DNA sequencing machines capable of generating tens to hundreds of million sequence reads in a single run at a modest cost have transformed functional genomics. It is now simpler, more accurate and in many cases less expensive to analyze the composition of complex nucleic acid mixtures directly, by sequencing, than indirectly by hybridization to a DNA microarray, as had been the norm for the last decade. High-throughput sequencing has also enabled new functional genomic experiments that could not be efficiently carried out with microarrays. The Vincent J. Coates Genomic Sequencing Laboratory at the University of California, Berkeley has catalyzed this shift by providing Berkeley faculty with access to next-gen sequencing, along with training and assistance in experimental design, sample preparation and data analysis. The center's two Illumina Genome Analyzers have been in constant use since they came online (one 15 months ago, a second 9 months ago). During that time, 23 NIH-funded (and 27 total) laboratories have used these machines to carry out a wide variety of functional genomic experiments, including: quantifying mRNA abundance, measuring mRNA synthesis and decay rates, identifying alternative splice forms and the frequency of their utilization, comparing the expression of different alleles, mapping the locations of regions bound by transcription factors and other DNA interacting proteins, determining the genome-wide distribution of epigenetic marks, cataloging the RNAs bound by RNA binding proteins, pinpointing mutants and genotyping individuals from large crosses. Analyzed samples have come from multiple species of bacteria, protists, plants, fungi and animals, including both model and non-model systems, mouse and human stem cells, and normal and diseased human cell-lines and tissues. In addition, Berkeley investigators have developed new sequencing-based techniques to characterize the in vitro affinity of DNA binding proteins to their targets, and to rationally evolve proteins to have desired functions. The center has been used to sequence several bacterial, fungal and animal genomes. Supporting this flurry of activity is a talented group of computational biologists at Berkeley developing innovative methods for base-calling, read mapping, assembly and analysis of this high-throughput sequencing data, and distributing and supporting software implementation of their methods that are actively used at Berkeley and elsewhere. With reasonably stable experimental protocols and analysis methods, the major factor limiting the wider utilization of this technology at Berkeley is sequencing capacity. Our two machines operate continuously and produce an average of 10 runs per month per machine (the length of a run depends on the desired read length and paired end status). Nonetheless, the queue of samples gets longer and longer, and there is currently a delay of nearly two months to have samples processed. We are requesting funds to purchase an additional Illumina Genome Analyzer IIx DNA sequencer to serve the demonstrated need of the Berkeley biology community. We have a fully operational facility, with an outstanding director, that has been producing high-quality data from Illumina sequencers for over a year. A new machine would be put to immediate use, and would have a significant positive impact on NIH-funded research in our community.