. 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.
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