Frozen DNA should be sent to:
Please ensure that a copy of the batch paperwork is enclosed in the package.
Please contact Alison Brown (617-768-8470) for more information.
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 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 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.
There are also several validated pre-manufactured panels for Illumina GoldenGate Genotyping Assay. Up-to-date information on these panels can be found at Illumina’s website.
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. It is recommended that DNA be stored in TE buffer (10 mM Tris, ph7.5: 1 mM EDTA). Full-skirted 96-well plates, such as AB0800 from Fisher Scientific are the only well plates accepted. Please leave well H12 empty on each plate so we can add our internal control DNA to that well. It is recommended 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.
Submit SNPs to Alison Brown as rs numbers or sequence.
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 are the OmniExpress (700,000 markers), Omni2.5-8 (2.5 M markers), Omni5 (5 M markers) and the Exome targetted chip with 12,000 marker for genotyping and copy number analysis. 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.
20 ul Minimum volume of genomic 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. It is recommended that DNA is stored in in TE buffer (10 mM Tris, ph7.5: 1 mM EDTA). Please provide samples in 96-well plates, containing 94 samples. Please leave well H12 empty for internal controls to be added. We advise that our customers include duplicate samples as controls in to their DNA plates, to monitor quality of our work. Full-skirted 96-well plates, such as AB0800 from Fisher Scientific are the only well plates accepted. WGA DNA is not recommended due to decreased call rate and amplification of allelic bias present in the original WGA sample.
All DNA plates delivered to the lab for Infinium Genotyping will be quantitated by picogreen to ensure the concentration of the DNA is at the required 50 ng/ul. Each DNA plate will also be monitored for degradation by running a small subset of samples on a gel. Those samples with lower concentrations or high amounts of degradation will only be genotyped with permission from the PI with the understanding that PCPGM is not responsible for drop in data quality.
For QC, each batch of 95 samples will be genotypes alongside a positive control sample. The resulting genotypes from the positive control sample will be compared over time to ensure reproducibility and performance of our lab process.
Submit biallelic SNPs to Alison Brown as rs numbers or sequence.
This facility offers Methylation Analysis from genomic DNA or bisulfite treated DNA using the Illumina HumanMethylation27 and the Illumina 450K Infinium Methylation BeadChip (Available Nov 2010). The Illumina HumanMethylation27 analyzes 27, 578 CpG loci in 14,475 consensus coding sequences in NCBI database (build 36). The Illumina 450K Infinium Methyaltion BeadChip will offer coverage of all designable RefSeq genes, both in 5’ and 3’ regions.
Prior to methylation analysis. Genomic DNA must be bisulfite treated to convert unmethylated cytosines to uracil, leaving 5-methylcytosine (methylated cytosine) intact.
The Illumina Methylation assay amplifies bisulfite treated DNA by Whole genome amplification, which results in the conversion of uracil residues to thyamine residues, while 5-methylcytosine remains as a cytosine residue. Methylation status is then interrogated by binding of the fragmented, amplified bisulfite-converted DNA to 50-mer allele specific oligonucleotides attached to beads on a solid microarray support: For each locus there are 2 bead types, one interrogating 5-methylcytosine and one for unmethylated cytosine. This is followed by single base extension with labeled didoxynucleotides: ddATP, ddUTP and ddGTP are labeled with DNP, while ddCTP is labeled with biotin. Immunochemical staining then scanning of the microarray measures the intensity of the 2 dyes corresponding to methylated or non-methylated cytosine bases.
Raw data is delivered via GIGPAD in the form of a .csv file which connects sample ID to the methylation loci and a Beta value. The Beta value is a quantitative measure of DNA methylation, and ranges from 0 for unmethylated to 1 for methylated.
If bisulfite treatment is required, please provide a minimum of 2 ug genomic DNA at 50 ng/ul in a skirted 96-well plate, such as AB0800 from Fisher Scientific.
If a sample has already been bisulfite treated then 1 ug bisulfite-converted DNA in 20 ul elution buffer is required, per sample. Please be aware that bisulfite treated DNA is not stable and cannot be stored indefinitely, Zymo protocols recomment storage at –20C for no longer than 1 month. Please add only 94 samples into a skirted 96-well plate such as AB0800 from Fisher Scientific, we use wells A01 and H12 to add internal controls.
To ensure that bisulfite treatment has been completed all samples will be QC’d before hybridization. This is included in the price of the chip. WGA DNA is not suitable for methylation analysis.