SNP Genotyping method is determined by number of samples and SNPs to be targeted. Available platforms for custom SNP genotyping include Illumina GoldenGate and Illumina iSelect, Sequenom hME and iPLEX as the main approaches, with Taqman Allelic Discrimination and other methods as secondary techniques. Standard Panel SNP genotyping can be performed using Illumina GoldenGate, Infinium or Affymetrix Mapping Arrays. The facility can also perform custom fragment analysis for microsatellites, VNTRs and indels.
All customers must be aware that not all SNPs submitted for any of the Genotyping technologies can produce successful assays. Depending on the technology to be used, there can be a dropout rate of 5-20% of SNPs that fail assay design completely. This 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.
If it is possible to design an assay, there is then a further chance of the assay not generating good quality genotype data. Again the rate of drop-out at this stage is dependant on technology and this rate can be up to 25%. Because of this only those SNP assays that we consider to produce reliable genotypes will be delivered to the customer and subsequently charged for. Assays, which fail by our standards, will not be billed, unless the customer specifically requests that data.
With all genotyping, our goal is to achieve > 95% success rate in genotype calls for a SNP, with a minimum cut-off of >90%. When lower success rates are achieved, it is the user's choice as to whether to have the data and pay the costs for each genotype delivered, or to refuse it and pay nothing. One exception to this is that any monomorphic SNP assays will be charged for at the appropriate per genotype rate.
All services include assistance with SNPs selection and choice of platform for any particular project. Each project must be priced independently and a quote can be generated quickly for any investigator. Genotype results are provided to the user in electronic format via the HPCGG web portal GIGPAD.
The HPCGG Genotyping Service has been offering cost effective, high quality custom SNP genotyping using Sequenom MassARRAY Genotyping system for several years. Our 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. In this method, multiplexed PCR and then a minisequencing reaction are performed in a single well. The size of reaction products is determined directly by MALDI-TOF mass spectrometry, yielding genotype information. Specialized equipment for this work includes a Biomek multi-pipettor robot, a Multimek multi-pipettor robot, a nanoliter plotting robot for spotting the extension products onto chips, and the mass spectrometer. Quality control of Sequenom Genotyping is carried out on all projects by repeating the genotyping on at least 5% of the samples provides (usually one 96-well plate) and comparing the genotypes from both runs. We are unfortunately unable to perform QC on small projects where less than 384 samples are provided. In this case, it is recommended that at least 10% of samples be duplicated within the samples to act as QC.
Sequenom hME Genotyping
Sequenom hME Genotyping is the technique we have been using for large scale custom genotyping over several years. We are continuing to offer this method 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. 30 ul Minimum volume of genomic or WGA DNA is required at 1.25 ng/ul in skirted 96-well plates.
Pricing: Sequenom hME SNP Genotyping pricing
Sequenom iPLEX Genotyping
Sequenom iPLEX chemistry and software allows interrogation of up to 29 custom SNPs in one well, using minimal genomic DNA. This platform is suitable for researchers with projects containing 20 to 300 SNPs or insertion deletion polymorphisms.
In 2007 we have run approximately 2500 different SNP assays with 95% conversion of sequence to assay, 89% of which are at a level of 20-plex or over. Average genotyping completion rate over all assays is 96%. 30 ul Minimum volume of genomic or WGA DNA is required at 5-10 ng/ul in skirted 96-well plates.
Pricing:SNP Analysis
using Sequenom iPLEX Genotyping
The Illumina platform, the BeadStation 500GX, consists of a BeadArray reader, BeadStudio analysis software which can be used with a variety of chemistries for Genotyping, Gene Expression, Copy Number 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 Infininium 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
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 96 sample Sentrix Array Matrices (SAM's) or 16 sample BeadChips and alleles are read by fluorescent readout using the BeadArray reader.
15 ul Minimum volume of genomic or WGA DNA is required at 50 ng/ul if quantitated by picogreen. If quantitated by any other method then the concentration should be 75 - 100 ng/ul. We only accept full, skirted 96-well plates.
All custom SNP assay design is carried out using Illumina's SNP Knowledge Resource, which consists of a large SNP database and expert support service. 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.
There are also several validated pre-manufactured panels for Illumina GoldenGate Genotyping Assay Click on the links below to be taken to detailed information on the Illumina website
In the case of Illumina GoldenGate genotyping we recommend that at least 10% of samples be duplicated within the samples to act as QC, as no further QC of samples will be carried out in the lab.
The Infinium assay from Illumina allows whole genome genotyping at different levels of coverage using a variety of fixed content chips. We are happy to offer any chips currently available from Illumina, including the newest chips detailed in the links below
There are also available bovine and canine infinium fixed content BeadChips.
We are also happy to offer custom Infinium iSelect genotyping to interrogate up to 60K custom SNPs.
Pricing: Illumina Infinium pricing
Fragment Analysis using Capillary Electrophoresis
Due to high demand for Sequenom and Illumina Genotyping, we may not be able to perform fragment analysis in a timely manner, please contact Alison Brown at 617-768-8470 or abrown13@partners.org for more details
Single SNP allelic discrimination is carried out using the ABI 7900, which allows single-plex SNP interrogation over a large volume of samples. Pricing:
SNP Analysis using Taqman
Please contact Dr. David Hunter 617-525-2755 for further details.
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