Guidelines for Sample Preparation for Infinium Genotyping Assay DNA input

Infinium Assay Sample Input Guidelines

Sample input guidelines:

• Most genotyping assays require 200 ng of high-quality DNA (4 μl at 50 ng/μl concentration) ​​

• The LCG Quad assay requires 400 ng of high-quality DNA (8μl at 50 ng/μl concentration)

• GDA+ePGx assay requires 100 ng of high-quality DNA (5μl at 20 ng/μl concentration) ​​for each of the Amplify WGA and Amplify PGx plates

Any standard DNA extraction method or kit that provides high quality genomic DNA is suitable for Infinium Arrays.

DNA quantity* Determine DNA concentration using a double stranded DNA specific fluorometric method (eg, Qubit, Quant-it, PicoGreen) for accurate measurement of double-stranded DNA.

• DNA quantification results from UV spectrophotometric methods do not accurately reflect the concentration of double-stranded DNA and are not recommended.

• Using the quantification values from the Bioanalyzer or Tapestation is not advised.

DNA quality* The minimum recommended fragment size is 2 kb.

• Illumina does not provide DNA integrity (DIN) guidelines or cut-offs for DNA input into the Infinium assay. DIN values from a Tapestation or BioAnalyzer can be used to assess DNA quality, but are not required.

• The presence of RNA in samples does not interfere with the Infinium assay.

• UV spectrophotometry can be used to assess DNA purity and quality. Consider standard purity specifications:

  1. Protein/RNA free: 260/280 ratio >1.85

  2. Solvent free: 260/230 ratio >2.0

  3. Phenol free: no peak at 270 nm

EDTA is generally introduced through sample treatment or extraction method, and the presence of EDTA can affect the 260/280 and 260/230 ratio values. EDTA can affect enzyme activity during the initial amplification step of the Infinium assay, so it is recommended to reduce EDTA concentration or use Tris-HCl or nuclease free water as the elution buffer. For additional information, see Knowledge Base article DNA/RNA isolation considerations for Illumina Library Preparation Kits. The information in this article can apply to DNA input preparation for Infinium arrays.

Recommendations for processing low quality DNA in the Infinium Assay

FFPE samples generally yield highly degraded DNA and typically perform poorly in array-based applications such as whole-genome genotyping. Consider using the Infinium FFPE QC and DNA Restoration Kits, which were designed for processing FFPE samples but can also be used on DNA degraded for other reasons.

• The Infinium FFPE QC Kit:

• Uses a real-time PCR assay to evaluate the quality of prospective DNA samples.

• Determines whether the DNA samples are suitable for the restoration step.

• Is only compatible with human samples.

• The Infinium HD FFPE DNA Restore Kit:

  • Restores degraded DNA to an amplifiable state that can be used as input for the Infinium assay.

  • Is compatible with samples from any species.

For further information, refer to the following resources.* Technical Note: Analyzing FFPE Samples with Infinium Microarrays

• Search for Knowledge Base articles:

  • Infinium Assay Modifications for FFPE samples for Genotyping Arrays.

  • DNA Input Guidelines for Infinium MethylationEPIC Arrays.

For any feedback or questions regarding this article (Illumina Knowledge Article #2180), contact Illumina Technical Support [e-mail].