DNA sample quality and quantity specifications
Quick Overview
Note: DNAs are to be arrayed in 0.8 ml 96-well "Midi deep-well" plates that are provided by the Broad Institute CGA. These plates are given unique barcodes for tracking and quality-control purposes.
Platform |
Minimum DNA volume |
Minimum DNA concentration* |
Additional information |
|---|---|---|---|
| Sequenom iPLEX | 25 ul + 2 ul per pool |
5. ng/ul |
n/a |
| Sequenom hME | 25 ul + 2 ul per pool |
2.5 ng/ul |
n/a |
| Illumina Single use | 25 ul |
50. ng/ul |
|
| Illumina Multiple use | 60 ul |
50. ng/ul |
|
| Illumina Infinium | 40 ul |
50. ng/ul |
|
| Affymetrix | 20 ul |
50. ng/ul |
* DNA concentration as measured by PicoGreen method. Spectrophotometry at 260 nm (A260) is not a reliable nor recommended quantitation method for these platforms.
Platform-Specific Quality Specifications
Sequenom
There are no specific requirements for DNA quality.
Illumina
DNAs must be prepared in a concentration range of 50 to 100 ng/ul, as measured by PicoGreen.
All plates received by the CGA for the Illumina platform undergo DNA concentration measurement. Plates determined to contain samples whose concentrations are out of this range may be delayed in processing or returned to the investigator.
An Illumina project that uses a single panel of SNPs (one "OPA") requires 25 ul volume of each DNA.
A project that will use multiple SNP panels (up to six "OPAs") requires 60 ul volume of each DNA. This requirement holds for the Illumina linkage panel, which employs four OPAs.
An Illumina project that uses the Infinium fixed-content array (Hap317K and Hap550K) requires 40 ul volume of each DNA.
Affymetrix
DNA samples should be diluted to 50 ng/ul with TE with reduced EDTA (10 mM Tris, pH 8.0; 0.1 mM EDTA). Furthermore:
- DNA must be double stranded
- DNA must be free of PCR inhibitors
- DNA must not be contaminated with other human genomic DNA
- DNA must not be highly degraded
Recommended DNA extraction methods include:
- SDS/ProK digestion followed by phenol-chloroform extraction and Millipore Microcon(tm) or Centricon(tm) ultra- purification and concentration.
- QIAGEN QIAamp(tm) DNA Blood Maxi Kit.