Learn about normalization and replicate probe groups

When you create a microarray design for the CGH or Expression application type, eArray adds default replicate probe group(s) to your design. For the CGH application type, eArray also adds a default normalization probe group. Agilent offers the following information about these specialized types of probe groups:

Normalization probe groups (CGH microarrays only)

When you create a custom CGH design, it is important to ensure that the design contains content that enables accurate dye normalization during downstream data processing. The dye normalization step is used to correct systematic differences in the overall signal intensities between the red and green channels. The assumption is that these overall intensities should be the same. If your custom design fails this assumption, Agilent recommends that you add a normalization probe group to serve this purpose.

eArray selects an Agilent normalization probe group by default if one exists for the selected array format and species. If you do not use this probe group, you should include a set of probes that represent non-aberrant/non-variant regions for proper normalization. Ideally, these probes should cover all autosomes. For whole genome arrays, it is not necessary to include a specific normalization probe group since there should be a sufficient number of non-aberrant regions for proper normalization. The Agilent or user-defined normalization probe group should be used in Feature Extraction (FE) to properly normalize the array.

There is a size requirement for a dye normalization probe group. Its probes must occupy at least 1% of the total number of features on the array after filtering. If you do not use an Agilent or user-defined normalization probe group, and the assumptions of dye normalization are not met within reason, FE will not be able to correct all of the systematic dye bias. This will affect copy number estimates. For information on how to use the normalization probe group for normalization in Feature Extraction, see the section "View or change grid template properties" and follow the directions in "Change the default DyeNorm gene list" under "Browse file" in the Feature Extraction Software User Guide.

Why and when should I include normalization probes on CGH microarrays, and how many?

What is the Agilent normalization Probe Group? Does this Probe Group avoid known CNV regions or frequent aberration regions in common cancers?

Replicate probe groups (CGH and Expression microarrays)

When you create a custom CGH or Expression design, it is important to include a set of replicate probes. eArray selects a default Agilent replicate probe group if one exists for the selected array format and species. Agilent recommends replicating this probe group five times.

Replicate probes are used for calculating the QC metric Reproducibility in Feature Extraction and DNA Analytics. This metric is set to the median percent CV of background-subtracted signal for these replicate probes after outlier rejection. High scores for the Reproducibility metric usually indicate that the hybridization volume was too low or that the oven stopped rotating during the hybridization.

If you choose not to use this probe group, it is important to include a set of replicate probes for calculating these metrics. For user-defined replicate probe groups, Agilent recommends a minimum of 300 probes replicated five times. This is because Feature Extraction requires a minimum three times replication level after rejection of feature non-uniformity outliers (FNUOL). The default Agilent replicate Probe Group for the 1x244 K and 1x1 M CGH microarrays contains 1000 probes and should be replicated five times. The 1000 probes are a random selection from catalog arrays.

• Note: (Expression application type only)
  • The default replicate probe groups that are included in standard Expression arrays differ from those included in Exon arrays.
  
  • You can use an Exon probe group as a replicate probe group in an Exon array, but not in a standard Expression array.
  
  • You can use a standard Expression probe group as a replicate probe group in both standard and Exon microarrays.
  
  For more information on Exon microarrays, see Exon microarrays.