Agena Bioscience MassARRAY®

The Agena Bioscience MassARRAY® system is DNA analysis platform that efficiently and precisely measures the amount of genetic target material and/or variations and is suitable for a variety of research applications including Somatic Mutation Profiling, Genotyping, Methylation Analysis, Molecular Typing and Quantative Gene Expression (QGE). Detection by MALDI-TOF mass spectrometry (MS) offers high sensitivity and accuracy.

 

Creating a Mass Array Genotyping Submission

The UAGC is now accepting Agena (formerly Sequenom) MassARRAY submissions through our Laboratory’s Sample Lifecycle Manager (SLM) 

Prior to submitting, please contact Crystal Richt to discuss project details.

1. Download the Excel mag_SubmissionTemplateForm.xlsx and fill it in with your information.

2. Create Submission- Once you are logged in to SLM, click the "Create Submission" button on the "My Submissions" page

  • Select MassARRAY Genotyping  - mag

  • Select your lab group and select your account

3. Add sample IDs- You will then be asked to provide the sample IDs for your submission- select  “Submit Tubes” or “Submit Grid”

NOTE: It is preferable to receive samples in 96-well plates unless submitting to another service first.

  • If you are submitting samples in a 96-well plate (preferable), select “Submit Grid” and then paste in the 8x12 grid of sample names from an excel spreadsheet by selecting the option to “Paste Sample Map”. Please include only the samples (no headers) and leave empty wells without text, or put the word "empty" in those cells. Other text (including "blank") will be treated as a sample. 

NOTE: Please leave at least one space for a well labeled as “NTC” (No-Template Control) for every chip you intend to run. 

  • For samples arriving to the lab in individual tubes, select “Submit Tubes” and type in the sample IDs one at a time for each sample being submitted, or copy and paste from a list. 

  • Click either Submit Tubes/Submit Grid to finish the submission. 

You will then receive a tracking number; i.e. mag-15-04-20-CFDQ

4. Upload your filled-in mag_SampleSubmissionTemplate.xlsx file.

In the excel form, be sure to:

  • Record submission IDs in "Submission Info" tab, and fill out the remaining information in GREEN on that page.

  • Please view the "SNP_Group Template" tab for instructions on how to create your SNP group file. 

  • You can either modify the "SNP_Group Template" tab for your own SNP file, or create a new one in the "SNP Group" tab.

  • If you have additional information, quants for samples, or samples are in varying volumes, please provide the information in the optional "SampleList" tab.

After you create the SLM submission, on the submission page there will be multiple tabs across the top; select "Files" and then upload the filled-in Excel template from your computer. The UAGC staff will not be able to proceed with your order until they have the additional information from this form. If you are submitting multiple plates for one work-order, you only need to upload this file to one of the submissions.

If you have any questions, please email Crystal RichtYou can also call the MassARRAY Genotyping service directly at: (520) 621-0044.

What is MALDI-TOF?

Matrix-assisted laser desorption/ionization – time oflight mass spectrometry.

Agena Bioscience’s SpectroChip is coated with a matrix which allows crystallization of the PCR product on its surface. A laser is fired at the crystal which ionizes the molecules. These ions travel through a vacuum tube to an ion detector based on their mass. Smaller molecules travel faster than larger ones. Time of flight measures the difference in time different molecules hit the detector and the software calculates the mass of the fragments. MALDI-TOF can resolve mass differences of 16 Daltons.

 

SNP Genotyping

The MassARRAY® iPLEX Gold is the leading technology for SNP genotyping for sub-whole genome study applications. It is used for fine mapping and validation studies. Assays can be multiplexed from 2-36 SNPs per individual reaction. 

This service includes:

  • PCR amplification ~100bp fragment containing SNP of interest
  • SAP treatment-cleans up left over primers and dNTPs
  • Single-base extension reaction-generates fragments of different masses
  • Resin clean up-remove excess salt
  • Nanodispensing on SpectroChip
  • Data acquisition- detection and ratio analysis with MALDI-TOF mass spectrometer

 

 

Experimental Guidelines

This instrument can perform up to a 36-plex, but we typically target between 25 and 30 loci. As for any multiplex, the number of loci it contains depends on primer interactions, so we may not be able to design a full 36-plex. To obtain the largest multiplex possible, it is best to provide 40-50 loci with specific notes regarding their relative importance. Provide the SNP ID (rs# is fine) and the sequence. Within the sequence the SNP should be denoted in brackets (i.e. [G/A]), with 100-200 bases of flanking sequence on either side. Tri-allelic SNPs are also detectable. If there are other known polymorphisms within the sequence please denote them as X so we can design the primers around them. There cannot be polymorphisms bracketing the SNP of interest within 20bp. If there is a polymorphism on one side, we can design the primer on the antisense strand.

Sample Preparation

The DNA samples work best if they are eluted in water, RNase free or HPLC water is fine. The instrument is very sensitive to salt, so please avoid TE buffer. Each reaction requires 1uL of 10ng/uL DNA. The DNA can be submitted in 96-well or 384-well plates with at least 2 open wells for negative controls. It is best if 260/280 is between 1.7 and 2.0.

 

DNA Methylation Analysis

The MassARRAY® system can measure individual methylation ratios for CpGs within a target sequence. Relative methylation ratios can be assessed in a range between 10-90% with a standard deviation of 5%. The system allows for flexible experimental design and is scalable for the analysis of a few or several hundred regions over multiple samples. Long reads up to 600bp in one reaction enables discovery of differential methylation within large promoter regions.

Choose from pre-validated assays in the EpiTYPER panels. 

This service includes:

  • Bisulfite treatment followed by PCR with a T7 promoter tag
  • SAP treatment to clean up left over single stranded DNA
  • in vitro transcription with T7 Polymerase
  • Base-specific RNA cleavage in which the resulting cleavage pattern depends on the presence of methylated cytosine in the original genomic DNA
  • Resin clean up to get rid of extra salt
  • Nanodispensing on SpectroChip
  • Data acquisition with MALDI-TOF mass spectrometer provides quantitative analysis of methylation ratios

 

Experimental Guidelines

The system allows for flexible experimental design and is scalable for the analysis of a few or several hundred regions over multiple samples. Long reads up to 600bp in one reaction enables discovery of differential methylation within large promoter regions.

Select from pre-validated assays offered from Agena Bioscience. Use the EpiBrowser tool to browse genes and samples of interest, assess methylation status of CpG sites and view validated primer sequences for amplifying regions of interest.