We request funds toward the purchase of a PacBio RS Single Molecule, Real-Time, (SMRT) DNA sequencer. The specific advantages of the PacBio RS are: 1) no requirement for prior PCR amplification of sample DNA avoids associated problematic artifacts;2) long-range polymerization enables sequencing of regions containing repeats, de novo assembly, and RNA seq of alternatively spliced transcripts;3) direct detection of DNA modifications eliminates errors introduced by alternative chemical methods;and 4) potentially wide range of future single-molecule applications, including direct sequencing of RNA and direct monitoring of translation step times. The UCI Genomics High-Throughput Facility (GHTF) has been in existence for twelve years, providing custom medium format array, Affymetrix GeneChip array, and now Illumina format high- throughput sequencing to users within and outside of the UCI campus. Thus, it is an established base from which to introduce new sequencing technologies to the Southern California region. The PacBio RS is complementary rather than overlapping with the currently available Illumina technology in the GHTF, As a consequence of PacBio RS acquisition, twenty-five investigators would be directly enabled to perform projects in the areas of: 1) genome resequencing to identify bacterial virulence determinants that occur through DNA modification and recombination-mediated mechanisms;2) model organism genome resequencing to study the relationship between recombination, repeated sequences and single-nucleotide polymorphism (SNP)-related genetic variation;3) localized human haplotype determination to understand the relationship between cis-acting genetic variation and disease;4) direct sequence analysis of modified DNA to understand the relationship between modification and gene expression;4) long-range reads to determine alternative splicing patterns which underlie transcriptional regulation in development and disease;and 5) future applications to other single molecule reactions with fluorescent substrates, Funds have been committed by the campus to complete purchase of the PacBio instrument and extended warranty and to support a technician in the facility and a systems administrator for the bioinformatics pipeline. This is essentially an extension of the very successful, high throughput sequencing program initiated with the last NIH Shared Instrument Grant for the GAIIx sequencing platform. With that funding and cost sharing, the IGB informatics pipeline was established as was a Cancer Center Bioinformatics group and last year the Center for Regenerative Medicine cooperated with the GHTF to support a trade-up to the current Illumina HiSeq 2000, which is now in operation. In summary, the UCI Genomics and High-Throughput Facility is the ideal location in which to house this exciting new technology: trained staff, strong informatics, knowledgeable users and applications that are not met by current, more established technologies.
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