PacBio RS Single Molecule, Real-Time (SMRT) DNA Sequencer

描述（由申请人提供）：我们申请资金用于购买PacBio RS单分子实时（SMRT） DNA测序仪。PacBio RS的具体优点是：1)不需要事先对样品DNA进行PCR扩增，避免了相关的问题伪影；2)远程聚合可以对含有重复序列、从头组装和选择性剪接转录物的RNA序列的区域进行测序；3)直接检测DNA修饰消除了其他化学方法引入的错误；4)潜在的广泛的未来单分子应用，包括RNA的直接测序和翻译步骤时间的直接监测。UCI基因组学高通量设施（GHTF）已经存在了12年，为UCI校园内外的用户提供定制的中格式阵列，Affymetrix基因芯片阵列和现在的Illumina格式高通量测序。因此，它是将新的测序技术引入南加州地区的一个既定基地。PacBio RS与目前在GHTF中可用的Illumina技术是互补的，而不是重叠的。由于PacBio RS的收购，25名研究人员将直接能够在以下领域开展项目：1)基因组重测序，以确定通过DNA修饰和重组介导机制发生的细菌毒力决定因素；2)模式生物基因组重测序，研究重组、重复序列与单核苷酸多态性（SNP）相关遗传变异的关系；3)定位人类单倍型测定，了解顺式作用遗传变异与疾病的关系；4)对修饰DNA进行直接序列分析，了解修饰与基因表达的关系；4)远程读取以确定发育和疾病中转录调控的备选剪接模式；5)未来在荧光底物的单分子反应中的应用。学校已承诺提供资金，以完成PacBio仪器的购买和延长质保，并为该设施的技术人员和生物信息学管道的系统管理员提供支持。这本质上是一个非常成功的扩展，高通量测序项目启动与NIH共享仪器资助的GAIIx测序平台。有了这笔资金和成本分担，IGB信息学管道作为一个癌症中心生物信息学小组建立了起来。去年，再生医学中心与GHTF合作，支持对目前正在运行的Illumina HiSeq 2000进行升级。总之，UCI基因组学和高通量设施是容纳这一令人兴奋的新技术的理想场所：训练有素的员工，强大的信息学，知识渊博的用户和当前更成熟的技术无法满足的应用。

DESCRIPTION (provided by applicant): 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.