Thomas Jefferson UniversitySidney Kimmel Medical College

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Services

About Our Laboratory

The Jefferson Cancer Genomics Laboratory 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 next-generation sequencing component operates two Applied Biosystems 5500xl for extended genome, exome and RNA-sequencing including non-coding as well as ChIP-, RIP- and ClIP-seq. An Ion Torrent PGM is used for amplicon sequencing and gene panels resequencing.

The facility is dedicated to meeting the current and future needs of both researchers and clinicians and regularly implements new technologies and methods to provide the highest level of service. An integral role of the facility also is to test new equipment and assays in collaboration with biotech companies.

Computational and statistical expertise for design of genomics experiments as well as generation, management, analysis, visualization and dissemination of genomic data is provided by its bioinformatics component..

The Following Services Are Available

CLIA Certified

K-RAS
BRAF
Sanger sequencing
AmpliSeq Cancer panel (409 oncogenes and tumor suppressor genes)
Whole exome sequencing

Whole-Exome Analysis on the high-throughput SOLiD 5500xl sequencer (CLIA certified)
DNA amount:
      10mg
DNA concentration:
100ng/ml
Price per Sample:
$1200* (50+35 base reads)
$1800* (75+35 base reads)
DNA Copy Number profiling in cancer, using the Affymetrix OncoScan FFPE Assay Kit (CLIA pending)
DNA amount: 200ng DNA concentration: 5ng/ml Price per Sample: $3500
Whole-Transcriptome analysis using the Affymetrix HTA 2.0 GeneChip array
RNA amount: 150ng RNA concentration: 25ng/ml Price per Sample: $800*
Non-coding RNA profiling for human, mouse, and rat using the Affymetrix GeneChip miRNA 4.0 Array.
RNA amount: 500ng RNA concentration: 100ng/ml Price per Sample: $400*

*Price shown for KCC members

The Ion AmpliSeq Cancer Panel

The Ion AmpliSeq Cancer Panel includes 50 genes, which are linked to targeted anti-cancer drugs currently on the market. Tumor DNA is extracted from a formalin-fixed paraffin embedded (FFPE) tissue sample. Next generation sequencing (NGS) is performed to find variants and small insertions/deletions, including those present in small amounts of tissue and not detectable using traditional Sanger DNA sequencing. The results coupled with bioinformatics provide a detailed molecular profile of the tumor that includes a list of all mutations. The report, delivered within 14 working days, details the mutational signature and provides the treating oncologist with an evidence-based list of therapies currently on the market, which facilitates a targeted approach to treatment for a specific tumor genotype.

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Whole Exome Sequencing (WES)

Many cancer-causing variants and mutations are found within exonic, or protein coding regions, which are critical in the development of human cancers. WES enables a closer look at these regions by analyzing of all coding regions (exons) of the genome simultaneously using NGS-based techniques. Approximately 180,000 exons in the human genome (22,000 genes) are analyzed, including mitochondrial genome screening.  The test requires 200 mg of fresh frozen tumor tissue or 10 cc of peripheral blood. The report details the precise mutational signature of the cancer and is delivered within 6 weeks.

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For research only:

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

  • whole blood
  • buffy coat
  • buccal swabs
  • saliva
  • bacteria
  • human and animal tissue including mouse tails
  • formalin fixed paraffin embedded tissue
  • cell culture fluid and cells
  • plasmids
  • PCR-products
  • amniotic fluid
  • plants
  • mRNA extraction is also offered

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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.

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Microarray Analysis

Affymetrix GeneChip Human Transcriptome Array 2.0 (HTA 2.0) (Santa Clara, CA). HTA 2.0 provides the coverage of up to 285,000 full-length transcripts, which include 245,000 coding transcripts, 40,000 non-coding transcripts and 339,000 probe sets covering exon-exon junctions. Up to 6.0 million distinct probes on the array maximize exon coverage in order to measure all transcript isoforms (70% and 30% of the probes cover coding transcripts and exon-exon splice junctions and non-coding transcripts, respectively). This high-resolution array design requires 50 to 500 ng of total RNA input and is optimized for both FFPE and fresh frozen samples.

The OncoScan FFPE Assay kit, based on Molecular Inversion Probe (MIP), exposes genome-wide copy number and loss-of-heterozygosity (LOH), and the status of frequently tested somatic mutations with its enhanced resolution of ~900 cancer genes. The test recently released by Affymetrix identifies copy number changes associated with tumor characteristics and examines the association of amplification events with stage and grade of disease.

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Quantitative PCR

The ability to quantitate PCR products as a measurement of the amount of template which was present at the beginning of the reaction has been hampered by the need to make measurements in the linear phase of the reaction, between cycle numbers, which will differ even among identical reactions in different tubes. 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.

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Standard Sanger 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.

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Microsatellite Analysis

The 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.

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SNP/Mutation Detection & Analysis (Genotyping)

Numerous options for characterizing SNPs are available including TaqMan-based genotyping 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.

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.

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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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