Technology Overview
Pyrosequencing is a type of DNA sequencing based on the “sequencing by synthesis” method. Unlike traditional Sanger sequencing, Pyrosequencing does not require fluorescence dye labeling or gel electrophoresis, while allowing quantitative, real time detection of sequencing events. The Pyrosequencing platform is using the PSQ 96MA instrument to provide core services:
| Instrument | Vendors | Maximum # of Samples Per Run | Read Length |
| PSQ 96MA | Biotage-Qiagen | 88 | 50-60bp |
Principle Pyrosequencing technology allows real time detection of sequencing events using reliable chemistry and robust detection mechanisms. It uses an enzyme-cascade system, consisting of four enzymes and specific substrates to produce light upon nucleotide incorporation during DNA synthesis. The amount of light produced is proportional to the number of incorporated nucleotides. When light signal is detected, the base is registered and the next nucleotide is added.
Video: Pyrosequencing Principle
Applications
- Quantitative DNA methylation analysis
- Mutation analysis and genotyping
- Allelic quantification
- Microbial identification and drug resistance typing
- Pharmacogenetics
- Clinical research
- Hematology research
Service Charges and Ordering
Service Charges
Effective 1-Sep-2022
| Service * | Reagent Kit | # of Instrument Runs ** | Maximum # of Samples | HKU Academic Research | Other Academic Research |
| Low volume screening | 1 | 2 | 70 | HK$ 5,500 | HK$ 6,900 |
| Full plate screening | 1 | 1 | 88 | HK$ 4,000 | HK$ 5,100 |
- Experimental design discussion
- Guided usage of assay design software
- Troubleshooting and assay optimization advice
- Tutorial on data analysis software
Consultation charge at HK$ 270 per hour applies beyond the above 2 hours. ** Up to 5 assays can be accommodated per instrument run. Additional assay will be charged at HK$ 150 per assay. Samples for the same assay MUST be arranged together. *IMPORTANT:* To qualify for HKU pricing, the investigator must be a regular employee of HKU AND payment must be made from an internal HKU financial account that qualifies for internal transfer. Overhead charge is required for all incoming external funding.
For commercial clients, please enquire.
Service Ordering Please submit service request through iLab.
Workflow
Sample Preparation and Submission
Service Ordering Procedure
- Contact platform specialist for assay design
- Submit service request through iLab
- Attach Assay Setup Form and Pyrosequencing Sample Submission Form
- Prepare samples (See below for details)
- Submit samples and sequencing primer(s) to CPOS Genomics Core
Sample Preparation Guidelines
Prepare biotin-labeled PCR fragments (Sequence to be analyzed) according to the assay design.
The PCR primer that is complementary to the sequencing primer MUST be HPLC-purified and biotin-labeled.
Use no more than 0.2uM PCR primers with high number of PCR cycle to deplete excess biotin-labeled primers. Excess biotin-labeled primers will decrease assay efficiency.
The PCR products MUST be specific and clean (i.e. without primer dimers and non-specific products).
2-5 pmoles of PCR product are required per reaction. We strongly recommend quantifying of the PCR products by running a DNA Mass ladder in the same agarose gel.
Sample Submission
IMPORTANT: When multiple assays are included in the same run, samples for the same assay MUST be arranged together.
For low volume screening services, all remaining samples must be submitted within 6 months after the first sample submission.
Prepare 2-5 pmoles of PCR products in a normalized volume between 20-50 ul in a 96-well plate.
Load maximum 88 samples per plate in columns 1 to 11 only.
Seal the 96-well plate with adhesive film securely.
Label the plate clearly with your name and date.
Dilute the sequencing primer to 20uM and prepare at least 1.5ul per sample in 1.5 ml microtube.
Label the tube clearly with primer name and date.
Put the plate and tube(s) in clear plastic bag.
Data Collection
Due to low usage and limited vendor service support, the turnaround time may be extended.
Data will be delivered through our Online Data Delivery System (ODDS), notification will be sent to the email provided on the service request form upon data delivery
For runs with PSQ software, data will be exported into a MS Word file containing the Pyrograms, SNP calls or allelic frequencies.
For runs with Pyro Q-CpG software, the raw data files will be delivered.
Users can perform data analysis with the Pyro Q-CpG software installed in the user workstations at the CPOS Genomics Core.
Advance booking can be made through the Online Resource Booking System.
In-person software tutorial is available for first time user.
Please contact our platform staff.
Useful Links
For more information
Vendors’ external links for reference:
Contact
Ms KONG, Carol
Mr CHOW, Clement
