SEQUENCING & GENOMICS (Under Construction)



Cancer genomics - Paolo Fortina, MD, PhD, KCC, TJU.
The Cancer Genomics Shared Resource (CGSR) is a full-service facility. In the previous competitive renewal of the CCSG, the Nucleic Acid Shared Resource for DNA sequence analysis and oligonucleotide synthesis was assessed excellent to outstanding. Facilities include a Microarray Shared Resource, which serves primarily for preparation of mouse, rat, zebra, and human oligonucleotide custom arrays; KCC has also purchased an Affymetrix GeneChipTM system The Nucleic Acid Shared Resource expanded its services to include SNP genotyping on microarrays to fulfill the growing research need of KCC investigators and other TJU researchers. The new, shared resource provides a full array of services, from traditional DNA sequence analysis to microRNA and genome-wide SNP studies associated with differentiation and delineation of differences between normal and diseased states. Since its inception, the CGSR has linked seamlessly to the Biostatistics Shared Resource to support advanced analytical tools for genetic analyses.
Equipment includes an Applied Biosystems 3730 48-capillary electrophoresis system for DNA sequence analysis and microsatellite analysis, Applied Biosystems Model 7900HT with automated plate handler for high-throughput processing and an Applied Biosystems 7000 Real-Time Quantitative PCR System, both for SNP genotyping and/or quantitative RNA assays. 5 Applied Biosystems 9700 Thermal Cyclers are used for routine PCR and sequencing reactions. For multiplex SNP assays, the CGSR uses the Pyrosequencing PSQ HS 96A and the Beckman Coulter GenomeLab SNPstream Genotyping System. For genome-wide association studies and mRNA expression profiling, the CGSR utilizes the Affymetrix GeneChip System. For spotted microarray and microRNA-based experiments, arrays are constructed using the Genomic Solutions Gene Machine OmniGrid100 Microarrayer. The CGSR has a Perkin Elmer Scanner 5000 with ScanArray v. 3.1 array analysis software. RNA, DNA and dye-labeled probe quality control are monitored using an Agilent 2100 Bioanalyzer and Nanodrop ND-1000 with software v. 3.1.0. The CGSR houses a 96-well autopipettor to automate PCR cleanup routines and liquid handling tasks. The Informatics CGSR is responsible for managing the large microarray files for the CGSR and its users. A web-based delivery mechanism for result files, and a web-based order and account management system was created for the CGSR: the MicroArray Generated Genomic Information Exchange application, or MAGGIE. A virtual directory structure was devised that could span any number of physical disk drives, and could be accessed via a "drag-and-drop" display. This addressed the problem of the continual addition of storage for the high volume data output of microarray experiments.



DNA sequencing and genotyping - Bruce Kingham, DBI and Allen Biotechnology Laboratory, UD.
The Sequencing & Genotyping Center provides state-of-the-art genetic analysis for university research groups and outside users on a fee-for-service basis. Bruce Kingham is available for troubleshooting and consultation. The Center is equipped with the following instrumentation: The ABI Prism 3130XL Genetic Analyzer system is a fully automated, fluorescence-based capillary electrophoresis platform that simultaneously analyzes 16 samples. The system provides automated polymer loading, sample injection, separation and detection, and data analysis. The instrument can run unattended for up to 24 hours. The NanoDrop Technologies ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of extremely small samples with high reproducibility. The sample retention system eliminates the need for cuvettes and capillaries. Benefits include full spectrum absorbance (220-750nm) and small sample size (1-2ul). ABI GeneAmp PCR System 9700 Thermocyclers offer fidelity, accuracy, NIST traceable calibration, and fast response to temperature changes. Heating and cooling is thermoelectric, a heated lid provides for oil-free operation. Eppendorf Centrifuge 5804R & 5417: The 5804R is capable of high-speed centrifugation (up to 20,800 x g) of tubes and plates using interchangeable swing-bucket and high-speed fixed-angle rotors. Internal temperature settings range from -9C to 40C. The 5417, for microcentrifuge tubes only, has similar capabilities without refrigeration. Dell Optiplex GX270 workstation loaded with licensed software for sequence and fragment analysis. UVP Ultraviolet Transilluminator for gel imaging and documentation. The Illumina Cluster Station is a fluidics device that hybridizes samples onto a flow cell and amplifies them for later sequencing on the Genome Analyzer. It uses solid support amplification to create an ultra-high density sequencing flow cell with millions of clusters, each containing ~1,000 copies of template, in approximately 5 hours. The Illumina Genome Analyzer II sequences clustered template DNA using a robust 4-color DNA Sequencing-By-Synthesis (SBS) technology that employs reversible terminators with removable fluorescence. This approach provides a high degree of sequencing accuracy even through homopolymeric regions. High sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. The Illumina Paired-End Module directs the regeneration and amplification operations to prepare the templates for the second round of sequencing, generating >50 bp reads for a total of >10 Gb of paired-end data per run.



Biomolecular laboratory - Katia Sol-Church, PhD, Nemours/AIDHC.
The Biomolecular Core Laboratory (BCL) provides essential services in molecular biology and genetics to the Nemours clinicians and research staff and to affiliates researchers of UD and TJU. Under the direction of Dr. Katia Sol-Church, this core is staffed by Jennifer Holbrook, BS and Deborah Stabley BS. BCL supports services in automated DNA sequencing, DNA fragment analysis, SNP analysis; Real-Time PCR; custom oligonucleotide synthesis; robotic liquid handling methodologies; pyrosequencing and bioanalyzer "lab-on-a-chip" technology to more than 50 customers at Nemours and affiliated institutions. The availability of a low-cost, fast-turnaround, high-quality service is essential to many clinical and hypothesis driven projects within the Nemours Biomedical Research department. Operating under HIPAA regulations, the service core currently supports the Nemours Molecular Diagnostic DNA sequencing and fragment analysis CLIA-certified tests.