High-Throughput Polymorphism Detection Core

The mission of the High-Throughput Polymorphism Detection Core is to provide services to investigators conducting molecular analyses of somatic DNA collected as part of a wide range of investigations. This Core provides high-throughput assays of specific gene mutations and polymorphisms (SNPs) in the many situations where previously defined specific nucleotide alterations are of interest.

The OpenArray SNP Genotyping Platform is a nanofluidic chip based platform that allows us to run assays that we know work well using traditional 384- well format TaqMan assays. The OpenArray system allows users to run 16, 32 or 64 SNPs on one OpenArray. Depending on the number of SNPs on an OpenArray up to 144 samples can be run per OpenArray (in a 16-plex assay).

SNP Analysis using ABI Prism Taqman Allelic Discrimination Assay
Single SNP allelic discrimination is carried out using the ABI 7900HT, which allows single-plex SNP interrogation over a large volume of samples.

Taqman Submission Guidelines

SNP genotyping by MALDI-TOF mass spectrometry is performed using the Sequenom mass spectrometry system. 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.

This platform is most suitable for researchers engaged in large scale association studies: whole genome linkage mapping panels and large scale fine mapping panels. The genotyping is carried out in large multiplexes of between 384-1536 SNPs, in multiples of 96 SNPs, using Illumina custom SNP panels or standard validated pre-manufactured panels. The Golden Gate assay is an allele-specific oligo hybridization, ligation and extension assay followed by universal PCR amplification, allowing that no amplification bias can occur. These amplification products then bind to the 3 uM microbeads in 32-sample BeadChips and alleles are read by fluorescent readout using the iSCAN reader.

Custom SNP assay design is carried out using Illumina's SNP Knowledge Resource, which consists of a large SNP database and expert support service. The PCPGM Genotyping Facility is happy to help navigate this process for customers. This resource provides access to more than 1,000,000 high confidence, mapped, and annotated SNP markers and to validated SNP assays across the human genome. Customers should be aware that this process can take several weeks to complete.

The Infinium assay from Illumina allows whole genome genotyping at different levels of coverage using a variety of fixed content chips. The most recently released chips use 700,000 to 5 million markers for genotyping. The Genotyping Facility is happy to offer any chips currently available from Illumina, including the newest chips detailed at Illumina’s website. This facility is also willing to offer custom Infinium iSelect genotyping to interrogate up to 60K custom SNPs.

The latest Infinium HD assay is a PCR independent assay. It uses hybridization of the loci of interest to 50-mer probes, stopping one base before the interrogated marker. Single base extension is then carried out to incorporate a labeled nucleotide. Dual color (red/green) staining allows the nucleotide to be detected by the iSCAN reader and is converted to genotype during analysis with the GenomeStudio analysis software.

Raw data is delivered via GIGPAD and is in the form of a .csv file which connects sample ID with marker ID and genotype.

This facility offers Methylation Analysis from genomic DNA or bisulfite treated DNA using the Illumina 450K Infinium Methylation BeadChip covering 99% of RefSeq genes, including the promoter region, 5’UTR, first exon, gene body and 3’UTR regions.  After bisulfite conversion the process follows the Infinium HD assay detailed above.  Raw data is delivered via GIGPAD and is in the form of a .csv file which connects sample ID with CpG site and methylation status.

Please contact Alison Brown (abrown13@partners.org, Tel: 617-768-8470) for quotes and information on the Sequenom and Illumina platforms.

Website: pcpgm.partners.org/research-services

Sequenom Submission Guidelines

To use the Sequenom SNP Genotyping service, we require that you register at our secure site to submit DNA samples and SNP flanking sequence.

• To register to use this service
• Information on using the Sequenom SNP analysis system

Once you have registered and have had your application approved you can then go on to submit SNP flank sequence for assay design and DNA sample sheet information.

PUBLICATION ACKNOWLEDGEMENT: If research supported by this core facility results in publication, please acknowledge this support by including the following in your publication(s):

We thank Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the High-Throughput Polymorphism Detection Core, which provided __________ service. Dana-Farber/Harvard Cancer Center is supported in part by an NCI Cancer Center Support Grant # NIH 5 P30 CA06516.