SEQUENOM Genotyping

Time Frame

Processing time from beginning to end will vary. The actual laboratory process can take up to two days due to lengthy thermocycling and then additional days will be required for primer design, primer shipments, and data analysis.

Please contact us to discuss initial account set-up, primer designs, final costs, and complete time frame. Also, please contact us if you are interested in SNP analysis using MassARRAY Discovery apart from iPLEX applications.

We provide genotyping service from Monday to Friday. Results are available electronically.

Advantages

Some of the many advantages of using this technique are:

  • Accuracy : High quality data based on direct allele-specific mass detection.
  • Optimization : Robust chemistry yields successful reaction products and minimal troubleshooting and few errors.
  • Assay Design : Primer design software promotes optimal multiplex primer designs by rejecting erroneous designs such as those forming hairpins or primer dimers.
  • MassARRAY RT genotype-calling software calls each genotype in real-time eliminating a time-consuming process when processing thousands of assays.
  • Typer : Data analysis software is comprehensive and scientific with results provided in a variety of graphs incorporating Hardy-Weinberg Chi-Square and P-values.
  • Results : Delivered electronically as Microsoft Excel files or by CD available upon request.
  • Reproducibility : Experiments can easily be duplicated.
  • Production : High rate of SNP sequence converted to genotypes.
  • High Multiplexing : Up to 36 assays can be designed on 1 DNA sample.

Techniques

The MassARRAY SNP Multiplex Genotyping method is ideal for investigating 24-36 possible SNPs on hundreds to thousands of patients’ DNA samples. The process begins with a list of SNP sequences submitted for design to MassARRAY software. Amplicons between 80-120bp are targeted, and PCR and extension primers are designed providing the best primer sequences following strict design guidelines. Once primer designs are determined, they can be synthesized. All iPLEX primer concentrations are adjusted to ensure that multiple SNP allele peaks can be resolved on the MALDI-TOF spectra.

The iPLEX high-multiplexing SNP genotyping laboratory procedure for given DNA samples in 384-well microplate format:

  • Multiplexed PCR reaction : targeting ~100bp amplicon regions.
  • SAP treatment (shrimp alkaline phosphatase) : dephosphorylation of unincorporated dNTPs and PCR primers.
  • iPLEX reaction : multiplexed primer-based extension reaction occurring adjacent to each SNP site featuring mass-modified, universal termination mix.
  • Clean resin treatment : sample purification removing excess salt ion contaminants.

The final products, DNA solution containing unextended primers and multiplexed SNPs, are dispensed from the 384-well microplate onto a 384-pad silicon microchip using the MassARRAY nanodispenser. The mass of each SNP allele is detected on the MassARRAY Compact MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization – Time of Flight) mass spectrometer, and the results are analyzed with MassARRAY Typer 4 software.

Genomic DNA Preparation

  • Purification: Purify DNA using commercial kits. (Sequenom recommends PUREGENE Genomic DNA Purification Kit by Gentra Systems, Inc. which uses 10 mM Tris, 1 mM EDTA pH 7.0-8.0 and produces high yields of DNA per mL of blood).
  • UV spectrometer range: DNA wavelength must fall within A260-A280 = 1.7-2.0. Start from a large stock of genomic DNA at 50 ng/µl.
  • Concentration: 5-10 ng/µl
  • Volume: depends on the number of plex. Please provide 20 µl or more of each DNA sample for the reaction and for the possibility of reprocessing.

Minimum Number of DNA Samples

We request that you send us more than 96 samples per order.

SNP Sequence File

  • Create and save your SNP sequence file as a tab-delimited text file using Excel. (*.txt)
  • SNP ID: Give each SNP a unique name using alphanumeric characters (no more than 20 characters). Avoid problematic symbols and spaces (i.e. % # / ) Underscore, dashes, and periods are the only recommended symbols. For instance: ( tb_3021 or brca-48889 or xyz3.1234 )
  • You must include only 2 columns specifying the SNP_ID (the name of your SNP) and the SNP sequence with one tab space in between.
  • SNP sequences should have less than 500bp.
  • Example of SNP Sequence File:
SNP_ID SEQUENCE
X-9357 … GCGAGCCTACGTGA[C/T]GTTCTGTCCCA …
X8156 … CTCCAACCTAAAGGCGG[A/T]CCGAGCGATC …
Y_234 … CTTATGCCAAGTGTAGTANN[G/C]ATGACAACATTG …
1.543XY … CCTGACT[C/T]GGTACCGTGTGTTTAG …
  • SNP alleles of interest within the sequence are denoted by enclosed braces using a separator [C/T]. In between the braces, only use A, C, G, and T, – for an insertion [-/A] or a deletion [T/-]. The SNP allele should be typed in uppercase letters and should not have any spaces in between. However, you can designate lowercase letters for sequences to be avoided. All SNPs other than the SNPs of interest should be replaced with ‘N’. Designs with ‘N’ will be avoided by the software