Sequenom Genotyping

Sample Submission

Frozen DNA should be sent to:

Attention: Genotyping/

Alison Brown
PCPGM
65 Landsdowne Street
Cambridge, MA 02139
Tel: 617-768-8470
Fax: 617-768-8510
abrown13@partners.org

 

Please ensure that a copy of the batch paperwork is enclosed in the package.

More Information

Please contact Alison Brown (617-768-8470) for more information.

The PCPGM Genotyping Service offers cost effective, high quality custom SNP genotyping using Sequenom MassARRAY Genotyping system. This service includes primer and assay design through to production of genotypes. Currently post-production analysis services are not available. All steps involved in the process are highly automated and are tracked using a laboratory management system that includes bar coding.

This system can also be used to carry out allelotyping to measure the allele frequencies of SNPs in both pooled DNA and cDNA for allelic expression.

The Sequenom method is based on multiplexed PCR followed by a minisequencing reaction that produces an allele-specific extension product. These products are spotted onto chips, and flown in a MALDI-TOF mass spectrometer to measure the mass of the extension products. Automated allele calling is carried out in real-time to convert the mass of the extension product to an allele call.

Quality control of Sequenom Genotyping is carried out on all projects by repeating the genotyping on at least 5% of the samples provided by the customer and comparing the genotypes from both runs. The ability to perform QC on small projects where less than 384 samples are provided is currently unavailable. In this case, it is recommended that at least 10% of samples be duplicated within the samples to act as QC.

Customers should be aware that not all SNPs submitted can produce successful assays. There is a possible 5-20% dropout rate of SNPs that can be due to the sequence surrounding the SNP containing repeat regions, other SNPs in close proximity to the SNP of interest, at rich regions and runs of nucleotides next to the SNP of interest. Customers should also be aware that if an assay does pass assay design, there is a further chance that it may not generate high quality genotypes during production. Poorly performing assays are filtered out based on quality of the spectra and scatterplots produced to prevent poor quality data from being deilvered.

The goal of this research core is to achieve > 95% success rate in calls for a SNP, with a minimum cut-off of >90%. Please be aware that this number is achieved only when high quality DNA is submitted and can vary from assay to assay.

 

Sequenom hME Genotyping

Sequenom hME Genotyping is the technique used at PCPGM for large scale custom genotyping over several years. This method is offered as a service to researchers with projects containing lower number of SNPs or insertion deletions (1-20 SNPs). Multiplex SNP analysis of a maximum of 7 SNPs per plex is optimal. Sequenom hME Genotyping can be used alongside Sequenom iPLEX technique (see below) to enable genotyping of SNPs that cannot fit into a high level of plex using iPLEX assay design.

Sample requirements

30 ul Minimum volume of genomic or WGA* DNA is required at 1.25 ng/ul in skirted 96-well plates (such as AB0800 from Fisher Scientific).

SNP sequences for assay design

Please submit markers and sequences to Alison Brown for assay design. SNPs should be contained within brackets in the submission form and flanked by 100 bases on either side of the SNP. Please submit sequences in all capital letters when possible.

 

Sequenom iPLEX Genotyping

Sequenom iPLEX chemistry and software allows interrogation of up to 40 custom SNPs in one well, using minimal genomic DNA. This platform is suitable for researchers with projects containing 20 to 384 SNPs or insertion deletion polymorphisms.

Sample requirements

30 ul Minimum volume of genomic or WGA* DNA is required at 5-10 ng/ul in skirted 96-well plates (such as AB0800 from Fisher Scientific). 

SNP sequences for assay design

Please submit markers and sequences to Alison Brown for assay design. SNPs should be contained within brackets in the submission form and flanked by 100 bases on either side of the SNP. Please submit sequences in all capital letters when possible.

Note: While WGA DNA is accepted for sequenom genotyping, customers should ensure that the input DNA in to the WGA reaction should be of high quality and that a minimum of 10 ng of genomic DNA is used in as the source DNA. Failure to do so will generate poor quality genotyping.