Technology for genotyping
The Biobank Genomics Core at Personalized Medicine provides flexible, high-quality, high-throughput SNP genotyping to the Mass General Brigham research community. We offer Taqman genotyping as well as genome-wide methylation and genotyping assays on our Illumina iSCAN platform. We employ state-of-the-art automation, including Biomek FX, Perkin Elmer Janus, a QuantStudio5 Sequence Detection System and an Illumina iScan system.
For Taqman, all requested assays must be available on demand from Thermo Fisher, as we do not carry out assay design and optimization.
For Illumina Infinium, we stock the Global Diversity Array (GDA), Global Screening Array (GSA) and the Human Methylation 850 EPIC array. We can run any of the commercial arrays available from Illumina, with the exception of Infinium XT products. Please order reagents and arrays directly from Illumina and have the reagents shipped directly to our lab.
New chip types may require additional processing time for clustering and allele calling on completion of wet lab work. Please ask if TAT will be impacted.
Specimen requirements per technology:
Technology |
SNP Info |
DNA |
Concentration |
Quantitation |
Minimum Volume |
|---|---|---|---|---|---|
| Illumina Infinium Genotypes | Fixed Panels |
Genomic DNA Only |
50 ng/ul |
Picogreen |
20 ul |
Taqman |
RS# or customer-provided sequence information |
Genomic or WGA DNA |
5-10 ng/ul |
Picogreen |
30 ul |
Illumina Infinium Methylation |
Human/Mouse |
Genomic DNA or Bisulfite-Converted DNA |
50 ng/ul of Genomic DNA |
Picogreen |
40 ul |
The Illumina Infinium assay allows whole genome genotyping at different levels of coverage, using a variety of fixed content chips, such as the GDA and GSA, for genotyping and copy number analysis. Other chips can be ordered directly from Illumina and sent to us for processing. Please note that we are not able to process Infinium XT assays and are not able to contribute to the design and ordering of any custom content arrays.
The latest Infinium assay is a PCR independent assay. Whole genome amplification is followed by 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 in the form of a .csv file which connects sample ID with marker ID and genotype, a sample sheet, a GenomeStudio project, and the raw intensity files from the scanner (IDAT).
Specimen requirements:
Sample type |
Minimum quantity |
Concentration |
Buffer |
Container |
Controls |
|---|---|---|---|---|---|
Genomic DNA |
20 ul |
50 ng/ul by picogreen; 75-100 ng/ul if quantitated by other methods |
TE buffer (10 mM Tris, ph7.5: 1 mM EDTA) |
Full-skirted 96-well plate |
Leave well H12 empty for internal control DNA. We recommend including duplicate samples as controls. |
The Biobank Genomics Core at Personalized Medicine offers Methylation Analysis from genomic 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 five foot- and three-foot regions. Prior to methylation analysis, genomic DNA must be bisulfite treated to convert unmethylated cytosines to uracil, leaving five-methylcytosine (methylated cytosine) intact.
While we do not stock the Infinium Mouse Methylation BeadChip, we can use this assay. Please order directly from Illumina and ship to our lab.
The Illumina Methylation assay amplifies bisulfite-treated DNA by whole genome amplification, which results in the conversion of uracil residues to thiamine residues, while five-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 two bead types, one interrogating five-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 and scanning of the microarray measures the intensity of the two dyes corresponding to methylated or non-methylated cytosine bases.
Raw data is delivered 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 zero for unmethylated to one for methylated. Also included are a sample sheet, a GenomeStudio project, and the raw intensity files from the scanner (IDAT).
Specimen requirements:
Sample type |
Minimum quantity |
Concentration |
Container |
Controls |
|---|---|---|---|---|
Genomic DNA |
Two ug |
50 ng/ul |
Full-skirted 96-well plate |
N/A |
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 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 one month.
Quality control
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 Personalized Medicine 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 turnaround time. You may waive the option of quality control, 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.
For samples extracted outside of our lab, we suggest each plate contains 95 samples which 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.
Turnaround time
We work on a first-come, first-served basis. Our expected turnaround time depends on our queue at the time we receive your samples. Once receiving the reagents, processing takes two to three weeks for up to five plates. Larger projects may require additional processing time.
Ordering and pricing
Before genotyping can be carried out, please visit our order entry page to place a GIGPAD batch order. For quotes and customer inquiries, please email us.
