High-Throughput Polymorphism Detection Core (DF/HCC)
Director: de Vivo, Immaculata, M.P.H., Ph.D.
Location: A Unit of the Partners HealthCare Center for Personalized Genetic Medicine Facility, PCPGM, NRB 164, 77 Avenue Louis Pasteur, Boston, MA 02115
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.
Director: de Vivo, Immaculata, M.P.H., Ph.D.
Phone: (617) 432-1642
Role: Lab Manager, PCPGM Genotyping Facility
Phone: (617) 768-8470
|Facilities and Equipment:|
Location of Core: A Unit of the Partners HealthCare Center for Personalized Genetic Medicine Facility, PCPGM, NRB 164, 77 Avenue Louis Pasteur, Boston, MA 02115
- ABI PRISM 7900HT sequence detection system
- Beckman Coulter Biomek FX laboratory automation workstation
- Deerac Fluidics Equator nanopipetter
- Illumina iSCAN reader and Autoloader 2.0
- Perkin Elmer Janus liquid Handler
- Sequenom MassARRAY Analyzer
- Tecan Freedom EVO 150 liquid handler
- Custom Illumina GoldenGate genotyping
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.
- Genotyping service using Sequenom, Illumina GoldenGate and Infinium technologies
- Illumina Genotyping
The Illumina platform consists of an iSCAN reader with autoloader, a Tecan liquid handling system and GenomeStudio analysis software which can be used with a variety of chemistries for genotyping, gene expression, copy number analysis, methylation analysis and loss of heterozygosity studies. Genotyping can be carried out using either the GoldenGate or Infinium chemistry on large numbers of samples. Both GoldenGate and Infinium chemistries are based upon a proprietary microbead technology assembled into arrays with redundant bead types for increased confidence calls. The bead arrays are configured onto a wide variety of array surfaces depending on the assay being used. Fluorescent readout of the bead array allows identification of a particular SNP.
- Illumina Infinium genotyping
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.
- Illumina methylation analysis
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.
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.
- SNP Analysis using OpenArray Genotyping
SNP Analysis using Taqman OpenArray Genotyping:
The Taqman OpenArray platform allows users to run 16, 32 or 64 SNPs in parallel using Taqman reagents.
The per genotype cost varies with the number of samples and the number of SNPs. Please contact Hardeep Ranu for exact pricing information.
- SNP Analysis using Taqman
SNP Analysis using ABI Prism Taqman Allelic Discrimination Assay
Single SNP allelic discrimination is carried out using the ABI 7900, which allows single-plex SNP interrogation over a large volume of samples.
- Whole genome amplification service
DNA extraction, plating, and whole genome amplification.
|Departmental Web Link(s):|