> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/microarray/general/microarray-general-troubleshooting-list/000002747.md).
# How to interpret the Controls Dashboard for Genotyping projects in GenomeStudio
All Infinium BeadChips are equipped with a set of internal control probes. Controls help to identify samples for which data characteristics are significantly different and may need to be excluded as outliers from further analysis. Evaluation of the control probes can link possible root causes to specific steps in the assay protocol. Because intensity levels may be different for any given Infinium project, Infinium controls performance is not based on specific intensity thresholds. Instead, Infinium controls are evaluated based on relative intensities.
The figure below summarizes the key steps of the Infinium Genotyping assay and demonstrates where internal controls come into play. While individual controls can be particularly informative of certain steps in the assay workflow, they may still be affected by performance of other steps in the workflow. Therefore, built-in Infinium controls are most useful when assessed in combination with each other.
**Figure 1. Infinium workflow steps and their correlation with built-in controls.**
The Infinium assay controls are categorized into sample-independent and sample-dependent controls. Sample-independent controls evaluate BeadChip and reagent performance, efficiency of hybridization, and the staining process. The sample-dependent controls are used to evaluate sample quality and performance. The sample-dependent Stringency and Non-Polymorphic Control probes specifically target human DNA and are therefore not informative when working with non-human BeadChip products.
**Table 1. Built-in controls and their evaluation channels.**
| Control | Sample Dependency | Red | Green |
| -------------------- | ----------------- | --- | ----- |
| Staining | Independent | Yes | Yes |
| Extension | Independent | Yes | Yes |
| Target Removal | Independent | Yes | No |
| Hybridization | Independent | No | Yes |
| Stringency | Dependent | Yes | No |
| Non-Specific Binding | Dependent | Yes | Yes |
| Non-Polymorphic | Dependent | Yes | Yes |
##
## **Functions and expected outcomes of controls**
**Staining Controls (Sample-Independent)**
* Used to examine the sensitivity and efficiency of the staining step in the X-stain.
* Consist of beads covered with high or small (background) levels of dinitrophenyl (DNP) or biotin.
* Are directly labelled in successive rounds of adding green fluorescent strepavidin and red fluorescent anti-DNP antibody. DNP and biotin are directly attached to the beads.
* Do not depend on DNA hybridization to the BeadChip and do not require single-base extension.
\
**Figure 2.** **Expected behavior of Staining Controls and schematic representation of assay probes.**
**Extension Controls (Sample-Independent)**
* Test the efficiency of single base extension during the X-stain.
* Consist of hairpin oligos that function as both template and probe. During X-stain, probes are extended at the 3’ end using the probe strand itself as a template.
* Independent of DNA hybridization, but require successful single base extension and staining.
\
**Figure 3.** **Expected behavior of Extension Controls and schematic representation of assay probes.**
**Target Removal Controls (Sample-Independent)**
* Test the efficiency of stripping off DNA template after the extension reaction.
* The control target DNA, added with the hybridization buffer (RA1), is extended and labelled using the probe sequence as templates.
* Denaturation with 95% Formamide/1 mM EDTA (non-EX assays) or 60% DMSO + 1mM EDTA (EX assays) removes the labelled target DNA.
* Failure to remove the target DNA results in an increase in signal intensities over background levels.
\
**Figure 4. Expected behavior of Target Removal Controls and schematic representation of assay probes.**
**Hybridization Controls (Sample-Independent)**
* Test assay performance using synthetic targets instead of amplified DNA.
* The synthetic targets bind to complement probes on the array, and provide templates for single base extension.
* The synthetic targets are present in the hybridization buffer (RA1) at three levels, monitoring the response from high-concentration (5 pM), medium concentration (1 pM), and low-concentration (0.2 pM) targets.
* Appear in the green channel as signals with various intensities, corresponding to the concentrations at which targets are spiked into the assay.
* Require optimal stringency conditions during hybridization and washing of BeadChips.
* Depend on successful single base extension and staining.
\
**Figure 5. Expected behavior of Hybridization Controls and schematic representation of assay probes.**
**Stringency Controls (Sample-Dependent)**
* Test the stringency of the hybridization process.
* High stringency is achieved through increased temperature and optimized composition of the hybridization buffer.
* Perfect match (PM) controls are exactly complementary to their human DNA target, resulting in high signal intensities.
* Mismatch (MM) controls probes have mismatched nucleotides between target and probe to affect hybrid stability, and are expected to yield signals of much lower intensity under optimal stringency conditions.
\
**Figure 6. Expected behavior of Stringency Controls and schematic representation of assay probes.**
**Non-Specific Binding Controls (Sample-Dependent)**
* Monitor the specificity with which amplified DNA hybridizes to the BeadChip, assess sample DNA quality, and identify presence of non-human DNA.
* The probe sequences for Non-Specific Binding Controls are complementary to bacterial sequences and should not hybridize to human sequences under standard hybridization conditions.
* Loss of the specificity of the assay and binding of non-human sequences complementary to control probes are expected to lead to increased signal intensities.
**Figure 7. Expected behavior of Non-Specific Binding Controls.**
**Non-Polymorphic Controls (Sample-Dependent)**
* Test the overall performance of the assay, from amplification to detection, by querying a particular base in a non-polymorphic region of the human genome.
* Used to compare assay performance across different samples.
* One Non-Polymorphic control has been designed for each of the four nucleotides.
\
**Figure 8. Expected behavior of Non-Polymorphic Controls and schematic representation of assay probes.**
**Restoration (Sample-Independent)**
* Strong signal intensity is only expected if samples were treated with Illumina's Infinium HD FFPE DNA Restore Kit. Otherwise, data points are expected to be at background levels.
* Does not depend on the quality of sample DNA.
* Should show no activity for samples that have not been processed with the FFPE Restore kit.
* Uses a short oligo that has been spiked into the Infinium HD FFPE Restore Kit. Due to the chemistry of the Infinium HD FFPE Restore Kit, the control oligo will be available to bind to its complement on the BeadChip only if the restore process functions properly. Detection of reduced intensity of the Restoration Control may indicate that the DNA restoration process has been compromised.
* Detected in the green channel
**Figure 9. Expected behavior of Restoration Controls.**
External links: [Evaluation of Infinium Genotyping Assay Controls Training Guide](https://support.illumina.com/content/dam/illumina-support/courses/eval-inf-controls/story_content/external_files/Infinium_Controls_Training_Guide.pdf)\
Recorded Webinar: [GenomeStudio Genotyping: Evaluating Infinium Assay Controls](https://www.youtube.com/embed/MuDBayIegkg?autoplay=1\&rel=0)
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2747), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002747%20-%20Microarray%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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