Technology Overview
The Core is equipped with the MassARRAY facility. The MassARRAY system was created based on the technology of MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. This technology was seen as a fast method for providing reliable molecular weight information which was more informative and unambiguous compared to gel electrophoresis, and was shown to be an attractive alternative to analyze diallelic or trialleic single nucleotide polymorphisms (SNPs), point mutations and insertion/deletion mutations in amplified DNA fragments. The various applications of the system include SNP genotyping, DNA methylation, molecular typing, quantitative gene expression and somatic mutation profiling.
A range of high tech machinery are employed to operate the system, including a MassARRAY Analyzer 4 mass spectrometer, the MassARRAY RS1000 Nanodispenser, and a series of Applied Biosystems 384-well PCR machines.
A series of MassARRAY custom software are also incorporated into this system capable of designing SNP assays/primers and collection of SNP data. Using the MassARRAY online assay design software, SNP assays can be designed as multiplexes, where maximally up to 40 SNPs can be analysed simultaneously within a single well of a 384 well plate. This generates throughput in excess of 10,000 genotypes per day. CPOS provides iPLEX Pro assay for service now (as its previous reagent version, iPLEX Gold assay, is no longer available in 2022).
Service Charges and Ordering
Service Charges
The MassArray Genotyping service is charged on a per chip basis which covers reagents and consumables for iPLEX Pro assay (primer cost excluded). In the most ideal case, one chip can be used for 356 samples x 36 SNPs (max).
Service charge will depend on sample size and SNP number. Please enquire.
Primer Cost
Primer scales will depend on sample size and SNP number. The Core will help identify the most cost-effective ordering plan after project confirmation. High quality primers from validated oligo supplier are to be ordered via the Core in order to ensure downstream data quality.
3 assay designs are included for each job order, i.e. the initial SNP list can be modified twice for re-design with no extra cost. If more than 3 assay designs are necessary, assay design fee will be charged. Please contact us for details. A HK$ 500 assay design fee will be levied should the assay design is not confirmed for proceeding within 3 months.
Service Ordering
Please submit service request through iLab.
Sample Quality Requirement
DNA samples MUST be:
- double-stranded
- free of PCR inhibitors, e.g. heme (from blood), EDTA and salts
- pure without contamination of any other genomic DNA
- with A260/280 ratio between 1.7 and 2.0
- intact (~10-20 kb) as shown on 1% agarose gel with proper size marker
Workflow
Project Initiation and Assay Design
Initiate a project at the Core
Email the following information to platform specialist (massarray.cpos@hku.hk):
- Institute
- Department
- Name of Principle Investigator (one responsible for payment)
- Project name
For new Principle Investigator, please also provide billing address and email address.
After complete information is received, users will be provided with an assay design quick guide for preliminary assay design.
Preliminary assay design by users
Choose target SNPs from database, e.g. NCBI dbSNP.
Follow instruction in the assay design quick guide provided to perform preliminary assay design with Assay Design Suite on Agena website.
For NEW users, please contact us for Agena account registration.
Submit Assay design files
Export all results in zip format from Assay Design Suite. Submit service request through iLab and attach the zip file for assay design review.
The result zip file should include:
- Assay design: contains assay information including grouping, primer sequences, PCR parameters etc.
- Plextend: contains amplicon sequences of each SNP.
- Prextend: contains primers validation information
- Proxsnp: contains proximal SNPs information in assay design input files
- Sequence Retrieval: contains SNP sequences
If users apply special setting (e.g. prioritizing SNPs) for assay design, please let us know when emailing the result zip file to us.
The Core will review the assay design and user will then receive reviewed assay design file (xls) for their final confirmation.
Finalize assay design and order primers
- Check reviewed assay design file (xls) received from the Core and confirm groups to be proceeded.
- With your confirmation, the Core will order primers on your behalf in specific format.
Sample Preparation
Sources of Genomic DNA
- Whole blood
- Buffy coat
- Cultured cells
* WGA DNA can also be used for iPLEX Pro. However, high quality starting DNA is required. Please note that the resulting clusters may not be as tight as that would be observed with genomic DNA.
Genomic DNA Extraction/Purification Methods tested at Agena
- PUREGENE™ Genomic DNA Purification Kit (Gentra Systems, Inc) on whole blood
* Genomic DNA may be isolated using any method you wish. The only requirement is that whatever method you use yields highly pure DNA suitable for PCR.
Sample Submission
* Note that only 89 wells per 96-well plate are utilized for samples as 7 wells are used for controls.
* In order to maximize the capacity and also be cost effective, users are recommended to submit samples in multiple of 89.
* Please assign unique sample ID to every sample.
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- Determine sample concentration and A260/280 ratios by UV absorbance. Make sure the concentration is between 10 – 20 ng/uL and A260/280 ratio 1.7 – 2.0.
- Run 4 duplicate check controls and 6 randomly selected samples for EACH DNA sample plate on 1% agarose gel along with proper size marker (see acceptable gel picture example below).
- Download “plate submission form”.
- Type in sample ID, sample concentration and OD measurement into the worksheet “plate format (Import by List)”.
- Check Plate format, name duplication and the number of samples that pass/fail sample requirement in the respective worksheets.
- Submit the file as attachment through iLab.
- Label on the side of a 96-well PCR plate with your name, date and plate ID.
- Transfer DNA samples in the 96-well plate according to the guide below:
- Seal plate tightly with good adhesive film. Make sure no leakage.
- Call platform specialist to arrange sample submission time.
- Submit gel photos (labeled clearly with sample ID) as attachment through iLab.
- Bring the sample plate(s) to the Core.
Example of an acceptable gel photo
Data Collection
Data Delivery
Upon completion of experiment, data undergoes quality control (QC) and review by our fully trained platform specialists.
User will receive an email containing 5 files:
- Plate check report (xls): quality check report for each experimental plate. Provides information such as call rate, number of SNPs passed or failed, and concordance check for duplicate controls and NTC.
- Genotype file (xls): lists all genotypes for each sample of each SNP with CPOS assay ID.
- Genotype rsNo (xls): lists all genotypes for each sample of each SNP.
- Assay design file (xls): updated assay design file with CPOS assay ID.
- Genotype transpose file (xls): lists all reformatted genotypes for each sample of each SNP.
Please secure your own copy of data. The Core will not be responsible to keep your data indefinitely.
Processing Time
Time for completion of service may vary depending on the number of samples submitted, number of assay groups to be done and the level of service demand at sample submission.
Turn-around time will be informed at time of sample submission.
Samples will be put in a processing queue as soon as the samples are all ready.
In case of unforeseeable delay, users will be contacted immediately.
Terms of Service
Platform limitations
Please note that there are some limitations of every platform for different technologies. Below lists the possible factors which may lead to lower call rates for some assays.
- This platform incorporates high throughput technology, only one PCR condition is utilized which may not perfectly optimize all primers in a multiplex reaction.
- On rare occasions with some assays, the MALDI matrix (sample spot on a SpectroCHIP) may not form properly during nanodispensing affecting the application of MALDI-TOF mass spectrometry.
Sample Quality Issues
There is a pilot check during sample processing.
If your samples failed in the pilot check, you will be notified and offered two options:
- continuation of the experiment with the consent of releasing the Core’s liability in final data quality
- termination of sample processing, in which the cost of pilot check will be charged
Samples, once processed, will be subjected to full service charge.
Special notes
- The Core has no obligation for failure of experiments (e.g. contamination, genotyping error or low call rate) if users fail to follow the instructions given and/or provide insufficient information to our staff.
- Users have the responsibility to perform quality control on their DNA samples. The Core has no obligation for failure of experiments (e.g. contamination, genotyping error or low call rate) due to sample mix up and/or degradation of DNA samples.
- Users should follow the existing queue of MassArray genotyping service. Jumping queue is strictly prohibited.
- Further amendments of assay design are NOT permitted after the email confirmation of primer ordering is sent by users. Users are requested to submit a new job thereafter.
Leftover Samples and primers
- After the completion of the experiment, sample plates and primers will be kept for 30 days.
- Users are welcome to collect any leftover materials during this period, after which, they will be discarded without notification.
- Please contact platform specialist for pick-up arrangement.
Useful Links
The Agena Bioscience website is recommended for further technical information.
SNP database resources:
Contact
Ms FUNG, Joyce
Ms LI, Rachel
