Services and Personnel
Dr. Celia Chang, a Ph.D. biochemist, leads the Genomics Facility with 17 years of experience. Dr. Chang has the primary responsibility for all facility functions including gene expression, SNP genotyping and CNV studies, genome sequencing using the Illumina NextSeq 500, and facility management. Dr. Chang is assisted by Dr. Shashi Bala, Ms. Sonali Majumdar, Ms. Tran Nguyen, and Ms. Sandy Widura with a combined 60 years of experience in genomic technologies.
Gene Expression Analysis
In response to Illumina’s discontinuation of human arrays by the end of 2016 and mouse and rat arrays in and before 2015, the Genomics Facility now uses Lexogen QuantSeq 3’mRNA-Seq Library Prep kits to prepare library and run on Illumina NextSeq 500. This kit is perfect for measuring gene expression, can be used for RNA from any species of interest and is the best alternative to expression Microarrays.
Illumina Next-Generation Sequencing (NextSeq 500)
Single Read, Paired End and Multiplexing Sequencing
- DNA Sequencing
- Whole-Genome Sequencing
- Exome Sequencing
- shRNA amplicon Seq
- RNA Sequencing
- Strand Specific mRNA-Seq
- Strand Specific total RNA-Seq
- Small RNA-Seq
- Methylation Seq
- Gene Expression Analysis: Lexogen QuantSeq 3’ mRNA-Seq library prep kit is used to generate Illumina compatible libraries. It produces one fragment per transcript which allows more accurate determination of gene expression and make it the best alternative to Microarrays.
Library preparations for all next-generation sequencing applications are provided by the Facility. User prepared libraries are also accepted. Please email the NGS Sample Submission & Analysis Form (Excel file), completing both the sheets in the file, and consult with Shashi Bala or firstname.lastname@example.org for questions regarding sample preparation and sequencing. A short description of the project must be provided with the samples.
nanoString nCounter Analysis System : Pre-selected or custom gene Panels
- Gene Expression
- Single Cell
- miRGE(miRNA and mRNA)
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 Sample Submission Form. The completed form must be emailed to email@example.com or Tran Nguyen, 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), 215-898-4521(fax).
For each sequencing run, investigators must provide samples in numbered or labeled 1.5 ml Eppendorf tubes:
- Double-stranded DNA: 0.5-1.0 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 DNA Sequencing Sample 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.
In most cases, sequences for DNA samples delivered by 11:00 am will be available the next day at 3:00 pm.
- Microsatellite analysis
Click here for the Microsatellie STR HID Sample Submission Form.
The AmpFlSTR® Identifiler™ PCR Amplification Kit is a short tandem repeat (STR) multiplex assay that amplifies 15 tetranucleotide repeat loci and the Amelogenin gender determining marker in a single PCR amplification. The combination of the 15 loci are consistent with several worldwide database recommendations.
Please submit 5 ul of DNA at 10ng/ul for 16 loci assay.
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 qPCR and primer design for gene expression 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.
- SNP Genotyping Analysis using TagMan assay
- C.bovis Detection. Please click here for a sample submission form.
- RNA and DNA preparation for all services
- 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 figures can be provided for grant applications with sufficient notice. Email Dr. Louise Showe, Scientific Director, with questions.