The Agena MassArray is a cost-effective mass spectrometry-based system for high-throughput SNP genotyping, somatic mutation profiling, and methylation analysis of DNA isolated from fresh, frozen, and FFPE specimens. The system is ideally suited to validate SNPs identified by next-generation GWAS studies in larger sample sets. In addition, the MassArray is capable of detecting somatic mutations with 5-10% frequency in the original tumor-derived DNA.
Please visit the Agena Website for additional information.
iPlex Pro Chemistry
The MassArray utilizes Sequenom’s iPlexPro chemistry for SNP genotyping and somatic mutation profiling. The initial step involves PCR amplifying the region spanning the SNP or mutation of interest using forward and reverse primers (80-120 bp amplicons). Subsequent treatment with shrimp alkaline phosphatase (SAP) eliminates unincorporated nucleotides. Extension primers (one for each SNP or mutation) are added to the amplified DNA template along with a thermosequenase enzyme and four different mass-modified dideoxynucleotide terminators. The extension primers are designed to anneal immediately next to the SNP or mutation of interest. During the cycling step, the thermosequenase adds one of the four terminators, which each have a different molecular weight. The reactions are then desalted and 8-12 nL is spotted on a 96-well SpectroCHIP II using the RS1000 Nanodispenser. The chips are fired on the Sequenom MassAnalyzer 4 MALDI-TOF mass spectrometer, where a UV laser induces desorption and ionization of the DNA follow by time of flight mass spec detection. (Heavier ions take longer to travel to the detector.) Based upon the initial molecular weight of the extend primer, the MassArray detects which terminator was added and the software assigns a call for each SNP or mutation.
Assays can be multiplexed by increasing the molecular weight of the extend primer. The MassArray is capable of detecting between 15-30 nucleotides with a molecular weight range of 5,000-10,000 Da. For germline SNP detection, a maximum of 30-40 SNPs can be multiplexed into one well. Somatic mutation profiling requires increased sensitivity and only 8-10 mutations can be multiplexed into one well.
The input DNA amount varies based upon how many wells per sample are required for the specific iPlexPro panel. The standard DNA input amount is 20 ng per well (2 µL per well of a 10 ng/µL DNA solution in water). The Molecular Diagnostics Laboratory will perform sample QC by measuring the DNA concentration using the NanoDrop. If the concentration is below the recommended amount, the investigator will be notified and given the option to proceed with the analysis. The Molecular Diagnostics Laboratory cannot guarantee that the analysis will be successful if the DNA quantity is below the recommended amount.
General Sample Preparation Guidelines
- Isolate DNA using a commercially available column purification kit (Qiagen, Sigma, etc.) and elute with water.
- Dilute DNA in water to a concentration of 10 ng/µL for iPlexPro.
- Avoid the use of TE or other buffers to minimize the generation of salt adducts.
- DNA should have a 260/280 ratio of 1.8-2.0.
- Please provide a minimum of 15 µL EXTRA per sample for QC and dead volume of our repeater pipettes.
- Only the recommended amount of DNA should be submitted as excess DNA will be discarded.
- DNA can be submitted in microcentrifuge tubes or 96-well plates.
- Submission in 96-well plates is required if your custom assay only uses 1-3 wells/sample (>24 samples to be run on a single plate).
- Requests should be made using GNomEx, and each sample should be labeled according to the submission instructions.
UltraSEEK Sample Preparation Guidelines
- Please follow the general sample preparation guidelines, except dilute DNA in water to a concentration of 1 ng/µL
- Use a fluorescence-based DNA quantification method such as Qubit PicoGreen
After firing the SpectroCHIP on the MassArray, the Molecular Diagnostics Laboratory staff will analyze the raw mass spectrometry data utilizing Sequenom’s TYPER Analyzer software. Based upon the mass of the unextended primer for each assay, the software will determine which terminator was added to the primer and will assign a call (A/T/C/G). If no DNA template was present or the initial PCR amplification step failed, only the unextended primer will be detected. The mass spectrum for each assay can be viewed using the Typer Anlayzer Software.
Example Mass Spectrum of a Somatic Mutation Assay
The Molecular Diagnostics Laboratory staff will perform an initial analysis of the data and generate a text file containing the frequency of each allele and call for every assay utilizing the TYPER Analyzer Software. For suspected SNPs or mutations, our scientists will view the mass spectra data to verify the call. If the call is a suspected false positive result, we will make every attempt to note our observations in the text file.
Both the text file containing the frequency data and calls as well as an “.xml” file containing the raw data will be uploaded to the experiment folder in GNomEx. To download the data, open the experiment folder in GNomEx, click the “files” tab, and select download files. The “.xml” file can only be opened with the TYPER Analyzer Software. The Molecular Diagnostics laboratory has a workstation with the software available for customers who would like to view and analyze their own data.
Due to the complexities of the iPlex Chemistry and mass spectrometry-based detection, the Molecular Diagnostics laboratory cannot guarantee the accuracy of the data and will not be held liable for false positive or negative results. We strongly encourage users to analyze their own data utilizing the TYPER software. In addition, we cannot guarantee every assay will yield useable data due to variables beyond our control, such as DNA quality, primer design, and proximal SNPs, etc.