Illumina Methylation Genotyping
The Translational Genomics Core at Partners Personalized Medicine offers Methylation Analysis from genomic DNA or bisulfite-treated DNA, using the Illumina Human 850K EPIC Infinium Methylation BeadChip. The Illumina 850K EPIC Infinium Methylation BeadChip offers 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 our LIMS (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.
|Sample type||Minimum quantity||Concentration||Container||Controls|
|Genomic DNA||2 ug||50 ng/ul||Full-skirted 96-well plate||N/A|
|Bisulfite-converted DNA||1 ug||In 15 ul elution buffer (per sample)||Full-skirted 96-well plate||Leave well H12 empty for internal|
WGA DNA is not suitable for methylation analysis. Genomic DNA will be bisulfite treated. To ensure that bisulfite treatment has been completed all samples will be quality controlled (QC) before hybridization
Please be aware that bisulfite-treated DNA is not stable and cannot be stored indefinitely; Zymo protocols recommend storage at –20C for no longer than 1 month.
All DNA plates delivered to the lab for Genotyping will be quantitated by picogreen to ensure the concentration of the DNA is at the required 50 ng/ul. Those samples with lower concentrations or high amounts of degradation will only be genotyped with permission from the principal investigator with the understanding that PPM is not responsible for drop in data quality. If concentrations are greater than required by the technology, then we are able to normalize the concentration in-house for a charge, or the plates can be returned to the customer for normalization. Please note that normalization will increase turn-around time. You may waive the option of quality control (QC), but then no guarantee is given to the quality of the genotypes, and all costs will be passed onto the customer regardless of quality of data. WGA DNA is not recommended due to decreased call rate and amplification of allelic bias present in the original WGA sample.
Each batch of 95 samples will be genotyped 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.
We work on a first-come, first-served basis. Our expected turn-around time depends on our queue at the time we receive your samples. Once receiving the reagents, processing takes 2–3 weeks for up to 5 plates. Larger projects may require additional processing time.