Core Center Resources:
Resources for Sequencing & Genomics
Cancer Genomics – Paolo Fortina, M.D., Ph.D., KCC, TJU
The Cancer Genomics Shared Resource (CGSR) is a full-service facility established over the past 10 months. 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. In addition, Developmental Funds were awarded to support a Microarray Shared Resource, which served primarily for preparation of mouse, rat, zebra and human oligonucleotide custom arrays. Since then, the Institution purchased an Affymetrix GeneChipTM system for use by KCC investigators. 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 University researchers.
To provide KCC members with flexibility of array options at the best price, the Cancer Center Committee determined that a centralized resource with a variety of platforms for genome-wide and gene-targeted service provision was needed.
The new, shared resource provides a full array of services, from traditional DNA sequence analysis to the most recent 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. This supports the most advanced analytical tools for genetic analyses. Software programs include GCOS v1.4 provided with Affymetrix GeneChip system, SAM, BRB array tools, custom-written, in-house SAS macros (for expression arrays), dChip, BRLMM Analysis Tool (Affymetrix SNP algorithms), CRLMM (alternative to BRLMM), and other publicly available software for SNP array analysis. In addition, NetAffx Analysis Center, GSEA and other pathway analysis software is used for more advanced analyses of expression arrays. This shared resource has been increasingly utilized since its inception.
The CGSR occupies approximately 1800 sq. ft. on the 10th floor of the Bluemle Life Science Buildings, in the Department of Cancer Biology. The laboratory has completely separated pre- and post-PCR rooms to prevent contamination.
Equipment
- 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.
- Five 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.
- Web log-ins provide investigator authentication and authorization so that researchers can only access and download their own data (Figure 1). Cumbersome FTP data transfers are avoided by using the standard web download mechanism. A similar web-based file delivery system also is used for delivering DNA sequencing results to the user. Software packages within the CGSR include Sequencer 4.7 from Gene Codes Corporation, SeqScape 2.5 and GeneMapper v.4.0 from ABI. The latter semi-automates the process of checking data for allele quality control, Mendelian inheritance and other processing of the data.
- SNP Evaluation Software Tool v.1.2 is provided for data analysis generated on the Pyrosequencing and Autoprimer v. 3.0 from Beckman Coulter, is used for fast, easy-to-use primer design support and tag assignments. Affymetrix package GCOS v. 1.4, BRLMM Analysis Tool 2.0 and GeneSpring GX 7.3.1 are available for array analysis and graphics.
- In addition to these informatics capabilities, the CGSR has a seamless link to the Biostatistics Shared Resource, which has most advanced analytical tools. As a general rule, users are readily referred to the Biostatistics Shared Resource for sophisticated analyses of data generated by the CGSR.
DNA Sequencing & Genotyping – Bruce Kingham, DBI and Allen Biotechnology Laboratory, UD
The UD DNA Sequencing & Genotyping Center is equipped with the following instrumentation:
- ABI Prism 3130XL Genetic Analyzer – The 3130XL 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.
- Apple G5 Server – The Xserve G5 offers dense power in a 1U server with dual 64-bit G5 processors at speeds of 2.3GHz. A UNIX-based server operating system with unlimited clients, up to 1.2TB storage, PCI-X expansion, dual Gigabit Ethernet, fast I/O ports and an optical drive.
- NanoDrop Technologies ND-1000 UV-Vis Spectrophotometer – The NanoDrop® 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 – High-performance 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 centrifuge 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 –9°C to 40°C. The 5417, for microcentrifuge tubes only, has similar capabilities without refrigeration.
- Dell Optiplex GX270 workstation – Computer workstation loaded with licensed software for sequence and fragment analysis.
- UVP Ultraviolet Transilluminator – Ultraviolet transilluminator for gel imaging and documentation.
The UD 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.
Biomolecular Laboratory – Katia Sol-Church, Ph.D., Nemours
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 the University of Delaware and Thomas Jefferson University. 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 strict HIPAA regulations, the service core unit currently supports the Nemours Molecular Diagnostic DNA sequencing and fragment analysis CLIA-certified tests.