The Partners Genotyping Facility, part of the Partners HealthCare Center for Personalized Genetic Medicine (PCPGM), provides flexible, high quality, high-throughput SNP genotyping to the Harvard-Partners research community, including Harvard Medical School, hospitals in the Partners HealthCare network, investigators in the Dana-Farber-/ Harvard Cancer Center, and the Harvard School of Public Health. The portfolio of Genotyping methods at PCPGM now includes Illumina, Sequenom, TaqMan and TaqMan OpenArrays.
|Amount of DNA per sample per plex|
|Sequenom||2-384 SNPs||Custom||Genomic and WGA DNA||5-10 ng/ul||OD||10-20 ng|
|Illumina GoldenGate||384-1536 in steps of 96 SNPs||Custom and Fixed||Genomic and WGA DNA||50 ng/ul||Picogreen||250 ng|
|Illumina iSelect||7K to 60K||Custom||Genomic DNA only||50 ng/ul||Picogreen||200 ng|
|Illumina Infinium||Up to 5 M SNPS||Fixed Panels (some chips allow custom additions)||Genomic DNA only||50 ng/ul||Picogreen||200 - 750 ng (depending on chip)|
|Taqman||1-8 SNPs||RS# or customer provided sequence information||Genomic or WGA||2ng/ul||Picogreen||4ng|
|OpenArray||16, 32, 64, 128 or 256 SNPs||RS# or customer provided sequence information||Genomic or WGA||50ng/ul||Picogreen||150ng|
TaqMan SNP allelic discrimination is performed on the ABI7900. In this method, specialized probe oligonucleotides are used to distinguish the SNPs during or at the end of a PCR reaction. Depending upon a number of factors, including previous implementation of the method for a particular SNP, this method will be the first approach for genotyping in some cases.
TaqMan OpenArray (formerly Biotrove) genotyping 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).
Sequenom genotyping is suitable for small numbers of SNPs, from 2 to 300 SNPs. It is performed using MALDI-TOF (Matrix-assisted laser desorption ionization - time of flight) mass spectrometry. This service includes primer and assay design through to production of genotypes. All steps involved are highly automated and are tracked using a laboratory management system with bar coding. Using the iPLEX chemistry, an average of 34 SNPs can be plexed into a single well of a 384 well plate, with a maximum of 40 SNPs per plex, while smaller SNP numbers can use the cheaper hME chemistry.
The Illumina GoldenGate assay 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 384 plex reactions then in multiples of 96 SNPs up to 1536 SNPs using Illumina custom SNP panels. Many standard panels are also available from Illumina.
The Illumina Infinium assay is also available for Whole Genome Genotyping. Genotyping is carried out on standard chips containing up to 5 million SNPs, at present. Custom genotyping of large numbers of SNPs is also available using the iSelect system. Several standard chips allow the addition of iSelect custom SNP sets to allow customization of the chip for the researchers specific aims.
The PCPGM Genotyping facility employs state-of-the-art equipment, including Biomek FX, Perkin Elmer Janus and Deerac Equator Nanodispensing robotic pipetting devices, a 384-well ABI 7900 Taqman machine, a chip spotting device, a Sequenom mass spectrometry system and an Illumina iScan system comprising of Tecan liquid handling and wash station, high-throughput iScan plate reader with autoloader. This high-tech equipment forms the core of the genotyping facility and enables throughputs in excess of 5 million SNP genotypes per day.