Evaluation of Infinium Staining Controls in the GenomeStudio Controls Dashboard

All Infinium BeadChips have built-in control probes designed to assess array and sample performance. While individual controls can be informative of specific steps in the Infinium workflow, they may be affected by other steps in the workflow. Therefore, built-in Infinium controls are most useful when assessed in combination with each other.

Considerations for evaluating Infinium controls

Infinium controls are evaluated based on relative intensities rather than absolute intensity thresholds. Signal intensities in the Infinium assay are subject to variations that are rooted in, but not limited to:

• Variability in DNA preparation methods, sources, or tissue types

• Operator variability

• Variability between scanners that do not impact results

• Inconsistencies in reagent storage temperature

These factors can manifest as batch-to-batch variability. Process controls are used to ensure that reagent, BeadChip, and scanner performance are within specification.

Sample-Independent controls evaluate BeadChip and reagent performance, DNA hybridization efficiency, and the staining process (XStain) of the Infinium workflow. The Sample-Independent controls are Staining, Extension, Target Removal, Hybridization, and Restoration.

Staining Control Chemistry

Staining Controls consist of beads covered with either high or low (background) levels of oligonucleotides bound to dinitrophenyl (DNP) or biotin (Figure 1).

Figure 1: Staining Control beads bound to DNP or biotin

On the BeadChip, the DNP on the control beads is directly stained with red fluorescence-labeled anti-DNP antibodies, which are counterstained with DNP-conjugated secondary antibodies (Figure 2). Similarly, biotin is directly stained with green fluorescence-labeled streptavidin, which is counterstained with biotinylated anti-streptavidin antibodies (Figure 2). Three rounds of staining and two rounds of anti-staining during XStain increases fluorescent signal intensities. The Staining Controls are then monitored in their respective channels, red (DNP) or green (biotin).

Figure 2: Fluorescence signal amplification of Staining Controls

Expected Staining Control Outcomes

Staining Control beads are manufactured with short oligonucleotides labeled with either DNP or biotin, while other controls are made with standard oligo-bound beads. Due to this distinct chemistry, Staining Controls perform differently than the other controls. Because DNP and biotin are directly attached to the oligos, Staining Controls are independent of both DNA hybridization to the probe and single-base extension. The expected Staining Control outcomes are summarized in Table 1.

Purpose	Name	Evaluate Green	Evaluate Red	Expected Intensity
Staining Red	DNP (High)	-	+	High
Staining Red	DNP (Bgnd)	-	+	Low
Staining Green	Biotin (High)	+	-	High
Staining Green	Biotin (Bgnd)	+	-	Low

Table 1: Expected control outcomes

• For the Staining Red controls, strong positive signals are expected in the red channel for the DNP (High) data points. The DNP (Bgnd), Biotin (High), and Biotin (Bgnd) signals are expected at background levels.

• For the Staining Green controls, strong positive signals are expected in the green channel for the Biotin (High) data points. The Biotin (Bgnd), DNP (High), and DNP (Bgnd) signals are expected at background levels.

• No comparison is made between the levels of positive signal in the red and green channels, nor is there a specific threshold value for expected intensities.

Troubleshooting

While all controls rely on successful staining, low Staining Control intensities do not necessarily indicate that the sample failed. If the other controls that rely on successful staining are as expected and the sample metrics* meet array specifications, low Staining Control intensities are not expected to impact data quality of the affected samples. Illumina recommends evaluating the controls in the Controls Dashboard in combination with each other, along with evaluation of the sample metrics, for a complete assessment of array and sample performance.

*Call Rate and Log R Dev scores for genotyping arrays, Detected CpG values for methylation arrays

Resources

For additional information on how to evaluate the Infinium Controls Dashboard, refer to the following resources:

• Module (30 minutes): Evaluation of Infinium Genotyping Assay Controls

• Recorded webinar: GenomeStudio Genotyping: Evaluating Infinium Assay Controls

• PDF: Evaluation of Infinium Genotyping Assay Controls Training Guide

For any feedback or questions regarding this article (Illumina Knowledge Article #6555), contact Illumina Technical Support [e-mail].