CANCER GENOMICS
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Kathryn Scott, B.S.
T: 215-955-9572
E: kathryn.scott@jefferson.edu
Adam Ertel, Ph.D.
T: 215-503-7452
E: adam.ertel@jefferson.edu
Sankar Addya, Ph.D.
T: 215-503-4673
E: sankar.addya@jefferson.edu
Stephen Dunn, B.A.
T: 215 503-6951
E: steve.dunn@jefferson.edu
Ellen Gutman, M.S.
T: 215-503-3977
E: ellen.gutman@jefferson.edu
Joan Kupper, B.S.
T: 215-503-3143
E: joan.kupper@jefferson.edu
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The Cancer Genomic Microarray Facility at the Kimmel Cancer Center was initiated in July 2006 to provide a full array of services, from traditional DNA sequence analysis to the most recent mRNA, microRNA and genome-wide SNP assay capabilities allowing the delineation of differences between normal and diseased states.
The facility comprises two central components: 1) the Microarray Laboratory, which provides instrumentation and expertise for nucleic acid extraction, quality assessment of total RNA and genomic DNA, RNA global transcription patterns, miRNA profiling, genome-wide SNP genotyping, mutation detection and DNA sequence analysis; and, 2) the Bioinformatics section, which undertakes all software development and implementation for data collection, analysis and management. Both of these components are run by research teams who provide training and support for collaborators/users at the KCC, TJU and elsewhere.
In order to maintain a strong position in the field, the facility periodically evaluates possible improvements to technology and development of new methods. An integral role of the facility also is to test new equipment and assays in collaboration with biotech companies.
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1. Nucleic Acid Extraction: DNA isolation and purification from human and animal matrices are performed daily using Chemagen's automation extraction robot and their proprietary magnetic bead technology. Using the chemagic magnetic separation module, DNA is extracted from many different biological matrices including but not limited to (1) whole blood (2) buffy coat (3) buccal swabs (4) saliva (5) bacteria (6) human and animal tissue including mouse tails (7) formalin fixed paraffin embedded tissue (8) cell culture fluid and cells (9) plasmids (10) PCR-products (11) amniotic fluid (12) plants. mRNA extraction is also offered.
2. RNA, DNA Analysis: The Agilent Bioanalyzer is used for rapid analysis of RNA and DNA samples. The system permits accurate sizing and quantitation of samples within minutes of loading and consumes only small amounts of material. This unit is especially important in assessing RNA quality and amounts prior to cDNA synthesis and labeling for microarray applications. In addition to characterizing total RNA used as starting material, the Bioanalyzer is able to check the quality of the RNA probe generated, check the status during labeling and fragmentation, and measure the amount which should be used in hybridization.
3. mRNA and miRNA Expression Profiling: The Cancer Genomic Facility provides microarray-based, genome-wide transcript expression profiling for definition of expressed as well as differentially-expressed genes in human, mouse, rat, Drosophila, yeast and zebra fish. The service is based on the Affymetrix GeneChip System. miRNA analysis also is performed using Affymetrix miRNA arrays or Luminex.
4. Quantitative PCR: Real-time quantitative PCR, using fluorescent probes and a detector such as the Applied Biosystems 7900HT, permits relative and absolute quantitation of transcripts with increased sensitivity, automatic data collection and multi-color capability. Subtle changes in transcript amount can be detected, and 96 or 384 samples can be run simultaneously, greatly facilitating gene expression studies.
5. DNA Sequence Analysis: The sequencing workhorse of the Facility is an Applied Biosystems 3730 Genetic Analyzer, a 48-capillary electrophoresis unit, which can perform hundreds of sequencing reactions per day. Runs in excess of 700 bases can be obtained in 1.0 hour and even longer runs can be obtained by varying run conditions. Plasmids, large insert clones and PCR products of a variety of sizes can be analyzed using BigDye Terminator Kit and cycle sequencing protocols. DNA sequencing-based services provided by the Facility include:
- Direct PCR product sequencing using either PCR primers or an internal primer
- PCR product purification and sequencing
- Direct sequencing of plasmid, cosmid, P1, BAC, PAC and other clones
- Plasmid/BAC/cosmid, etc. template DNA isolation and sequencing
- Entire clone insert sequencing using primer walking strategy
Recently, we have expanded our services to provide next-generation sequencing capability by implementing the AB SOLiD 3 System, an advanced genomics analysis platform that facilitates whole-genome DNA/RNA sequencing, tagged counting and discovery. The system is capable of yielding 25-30 gigabases of mappable sequence data, or 400 million tags, per run with an accuracy of 99.94%. It is anticipated that this new level of data output per run will continue to increase resulting in a further decrease the cost of operation. Multiple applications and projects are enabled with the SOLiD 3 System including: 1) DNA sequencing (whole genome and targeted); 2) RNA transcriptome and gene expression; 3) mutation screening; 4) SNP discovery; and, 5) chromatin occupancy and epigenetic studies (ChIP-Seq) associated with differentiation, delineation of differences between normal and diseased states. Therefore, within a few months, the Cancer Genomic Facility will be able to provide digital mRNA expression profiling from a single cell and from FFPE, measure alternative splice variants, perform for the first time genome-wide and targeted mRNA and microRNA-sequencing, DNA methylation bi-sulfite sequencing, determine LOH and copy number variation and perform massively parallel DNA sequencing.
6. Microsatellite Analysis: ThThe Facility provides microsatellite analysis using the ABI 3730 platform. The microsatellite assay detects microsatellite marker repeat lengths in genomic DNA using fluorescently labeled PCR reactions on the ABI 3730 sequencer. Mouse genotyping via marker-assisted profiling of selected informative microsatellite markers is available.
7. SNP/Mutation Detection and Analysis (Genotyping): Numerous options for characterizing SNPs are available including TaqMan-based genotyping, the SNPstream Genotyping system, and the Affymetrix GeneChip platform for high-throughput genotyping. Custom SNP panels can be designed for a particular project. Choosing the right platform starts with the list of SNPs to be tested and Facility team can aid in exploring custom multiplex options.
The Facility provides the TaqMan-based assay with readouts of the genotypes using the AB 7900HT instrument. Thousands of verified SNPs are available as reagents in which the PCR primers, conditions and allele-specific probes all have been verified (Assays-on-Demand™). Those SNPs not already included as "off-the-shelf" products can be ordered from the database and developed as verified reagents as well (Assays-by-Design™).
The Facility has made available the Beckman Coulter GenomeLab SNPstream Genotyping System for multiplex SNP genotyping in 96- or 384-well plates. Pre-formulated, ready-to-use SNPware Reagent Kits are optimized for robust single nucleotide extension (SNE) reactions and only 2 to 5 nanograms of genomic DNA are required per reaction. SNE results are automatically imaged from each well of a plate and processed into genotype calls. For genotype calling and data analysis QC, the software offers graphical interpretation of results, including cluster and Hardy Weinberg QC analyses, and additional view options.
In instances in which a genome-wide genotype is preferred, the Facility utilizes the Affymetrix 250K and SNP 6.0 GeneChips which provide the capability to assay large numbers of alleles in parallel. The array platform provides comprehensive genome coverage at high-resolution necessary for genome-wide association studies, loss of heterozygosity and gene copy number variation. The assay requires 500 ng genomic DNA as starting material.
We are currently implementing the TaqMan® OpenArray® Genotyping Instrument Platform capable to provide PCR-based SNP analysis of hundreds to thousands of samples and candidate SNPs. This all-in one solution provides efficiency and power through its ability to validate and screen up to 4,600 DNA samples in a single day with minimal hands on time and without the use of robotics. Using the system, one person can generate over 98,000 genotypes a day. The main components of the system are the TaqMan OpenArray Genotyping Plates (pre-loaded with pre-designed or custom TaqMan SNP Genotyping or Drug Metabolism Assays) and OpenArray technology, which utilizes a microscope slide-sized plate with 3,072 through-holes. The plates are arranged in 48 subarrays of 64 through-holes. TaqMan Genotyping Assays are pre-loaded and dried down in the plate and delivered ready for processing. Three TaqMan OpenArray Plates can be imaged simultaneously on the OpenArray NT Imager, providing high-throughput results. The system workflow requires thermal cycling and fluorescent imaging of TaqMan Genotyping Assays. Results are captured on a PC and the OpenArray Genotyping Analysis Software enables investigators to view specific assay data and scatter plots. The system is ideal for validating complex disease associations, cancer-associated markers or other studies requiring large sample sizes (e.g., ancestry linkage genotyping).
8. Luminex Multiplexing: The facility maintains a Luminex 200 instrument capable of assaying for a wide number of cytokines, chemokines and growth factors in human and animal fluids and tissue extracts. Commercially available targeted panels tailored to cancer, endocrine, cardiac, hepatic, gut are available for assay by Luminex. Using the principals of flow cytometry microspheres, lasers, digital signal processing and traditional chemistry, the instrument permits rapid, simultaneous assay of many substances with a minimum amount of sample. Often, a Luminex designed assay can take the place of many ELISAs reducing cost, saving time and sample. Additionally, the Luminex can be applied to measuring microRNAs using linked nucleic acid technology, HLA typing, cell signaling and transcription factors.
9. Bioinformatics Data Analysis: The facility offers standard analysis services for gene expression microarray data including differential expression analysis and cluster analysis. Advanced bioinformatics services are also available to provide contextual data analysis using tools such as GeneSpring, Gene Set Enrichment Analysis and Ingenuity Pathways. The application of these computational tools is tailored to provide comprehensive analysis to support studies using gene expression, genotype and genomic copy number data.
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