Genotyping Standard Operating Methods

Primer Design using SpectroDESIGNER

Description
SpectroDESIGNER is the assay design software supplied by Sequenom as part of the MassARRAY package.  This SOP describes how to design assays for running on Sequenom.

Materials

1. Tab delimited Text file from customer

2. Spectrodesigner software

Example: SNP input file

Method

Ensure that the file given by the customer is in the correct format. 

• The file should be a tab delimited text file.

• It should contain 2 columns, one headed “SNP_ID” and the other “Sequence”.

• Under SNP_ID the names of the SNPs should be no longer than 20 characters and should contain only letters, numbers and underscores.

• The sequence flanking the SNP site should be 100 bases on either side of the SNP and should contain no spaces or characters other than A, G, C, T and N.  

• All SNPs other than the SNP of interest should be replaced with “N”.

• The SNP of interest should take the following format [A/T], if the SNP is a one base insertion or deletion it should take the form [A/-].

• All characters should be in uppercase otherwise they will be ignored by the design software.

• Multiple SNP’s in one strand can be designed.

• The name the file is given will be come the group name of the SNPs, this name will be used as an identifier through assay processing.

 SpectroDESIGNER

• Open SpectroDESIGNER and select the “SNP group”, this is the file containing the SNP information described above.

• Select the level of multiplexing required.  The standard is between 3 and 8-plex.

• A terminator mix can be selected but for better assay design leave the default “Best”.

• The SNP capture parameters are as follows Minimum 60, Optimum 100, Maximum 120.

• The primary and secondary tags are hME-10 – this is the universal tail, which increases the mass of the PCR primers to over 9000 Da so that they will not interfere with the extension peaks.  They also help to standardize the primers so that they will work more efficiently in multiplex.

• The extend primer design parameters should be Min Tm 60, Max Tm 100, max Length 24 bp.

• When ready press run and assay design will commence.  When it is finished open the design summary file and view the assays.  Scroll down to see why certain assays have failed as it may be that they contain non-standard characters such as spaces.  

• Adjusting the order of the SNPs in the input file has the affect of changing which SNPs are multiplexed together.  This may mean that SNPs which dropped out the first time may work the second time around.

Safety implications

• None

Related MSDS

• None