Dr. Celia Chang, a Ph.D. biochemist, leads the Genomics Facility with 14 years of experience. Dr. Chang has the primary responsibility for Illumina gene expression, SNP Genotyping and CNV studies, genome sequencing using the Illumina GAIIx and HiSeq 2000, and facility management. Dr. Chang is assisted by Dr. Shashi Bala, Ms. Rajrupa Majumdar, Ms. Tran Nguyen, and Ms. Sandy Widura.
Gene Expression and Genotyping Studies
Illumina Bead Array Platforms
- Gene Expression Arrays (for human and mouse, including every step from RNA purification, amplification through hybridization and image quantitation)
- GoldenGate Genotyping Technology
- Infinium Whole-Genome Genotyping
- Gene Expression in FFPE samples
- Human promoter Infinium methylation studies from DNA purification and bisulfate conversion
Investigators are asked to choose the Illumina Beadchips that best suit their needs and provide information about the quantity and quality of the RNA being submitted. The average time required for hybridizations, starting with sample submission and ending with an image of the array, is 10 working days; however, this time will vary based on the numbers of samples and the quality and quantity of RNA or DNA.
The Sample Form (Word file) should be printed out and accompany all DNA/RNA samples submitted for analysis.
Samples submitted for analysis should be provided in eppendorf tubes clearly labeled with the following information:
- species and source of DNA/RNA sample
- purification procedure and whether RNA has been DNAse treated
- concentration of DNA/RNA
Nanodrop and Bioanalyzer analysis can be provided.
Please contact Celia Chang to schedule hybridization of your RNA or DNA samples.
Illumina Next-Generation Sequencing
Single Read, Paired End and Multiplexing Sequencing
- DNA Sequencing
- Whole-Genome Sequencing
- Exome Sequencing
- Transcriptome Analysis Applications
- Strand Specific mRNA-SEQ
- Small RNA-SEQ
- ChIP-Seq Analysis
All sample preparations for next-generation sequencing analysis are provided by the Facility. User prepared samples are also accepted. Please email the NGS Sample Submission & Analysis Form (Excel file), completing both the sheets in the file, and consult with NGS specialist for questions regarding sample preparation and sequencing. A short description of the project must be provided with the samples.
Capillary DNA Sequencing and Microsatellite Analysis
The facility uses an ABI 3130xl capillary machine for Sanger-type sequencing with fluorescently-labeled dye-terminators, the ABI four-color detection, and sequence analysis system. The ABI 3130xl is a 16 capillary machine that can sequence over100 samples/day. The 3130xl can also be configured for fluorescent fragment detection and microsatellite analysis and is equipped with Gene Scanning Software.
How to use the Sequencing Facility
Click here for the DNA Sequencing Submission Form. The completed form must be emailed to DNA_SEQ@wistar.org, and a hard copy must also be delivered with the samples.
Purified DNA is provided by the investigator with an order form specifying the quantity, form (double-stranded, single-stranded, or PCR product) and estimated size of the DNA. It is essential that the size and concentration of PCR products are determined accurately prior to sequencing.
For questions relating to the Sequencing Facility, please contact Ms. Tran Nguyen, 215-898-3991 (office), 215-746-7452 (fax).
For each sequencing run, investigators must provide samples in numbered or labeled 1.5 ml Eppendorf tubes:
- Double-stranded DNA: 1-1.5 µg of purified DNA at 0.2-0.25 µg/µl
- Single-stranded DNA: 1 µg of purified DNA at 0.1-0.125 µg/µl
- PCR products: at least 15ng of purified DNA per 100 bp of PCR product in 5ul water
A sample name and the primers to be used for each run must be clearly indicated on the submission form.
- M13 forward, M13 reverse, T7, T3, and SP6 primers are provided by the Facility at no additional cost. Specific primers must be supplied by the investigator at 3.2 pmoles/µl.
- Microsatellite Analysis: at least 450ng of DNA at 30ng/ul for 5 loci assay, and 5 ul of DNA at 10ng/ul for 16 loci assay.
In most cases, sequences for DNA samples delivered by 11:00 am will be available the next day at 3:00 pm.
What to expect from the Sequencing Facility
- For clean double-stranded templates, extremely accurate sequence reads of >=600 bases are obtained routinely.
- Text files of sequence data are e-mailed.
- The original result folder from the ABI base caller is placed in a computer folder for electronic retrieval if needed.
- Other modes of delivery can be arranged.
ABI Real –Time qPCR System
For miRNA profiling of human and mouse samples, digital PCR, Pathway specific PCR arrays, custom miRNA and gene expression arrays, ABI OpenArray NT Cycler System is used. For qPCR and primer design for microarray validation, ABI 7900HT Fast Real-time PCR System with 384 well plates is used. Where possible validated commercially available primers are used. Special primers may be designed by the user or by the facility.
- Protocols: RNA preparation, hybridization, RNA amplification
- Troubleshooting: Send an email with your questions to firstname.lastname@example.org.
- Training: By appointment only, hourly cost basis
- Grant applications: Core descriptions and Facility generated array figures can be provided for grant applications with sufficient notice. Email Dr. Louise Showe with questions.