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A control grid is a set of control probes that SureDesign adds to every microarray design. For every microarray design format and species, Agilent has a default species-specific control grid. These grids contain positive controls probes that were designed against the endogenous sequence for the species. Positive controls can be used for image orientation and to assess whether the sample was labeled. The “genomic” control grids are generic grids that contain only negative controls. Since they lack endogenous positive control probes, genomic control grids can be used for any species. The negative control probes are used to measure on element background.
Below are some of the probe types you can expect to find on a species-specific genomic control grid.
BrightCorner (e.g. HsCGHBrightCorner)
· Used for orientation purposes. These probes are placed in the corners of the array with a different pattern for each corner.
· These probes are designed to endogenous sequence and are thus species-specific. These probes are predicted to have high signal due to the multiple copies found in the genome.
DarkCorner
· Used for orientation purposes in the array corners. These, along with the bright corner probes, make up the corner-specific patterns.
· These probes are the same as the 3xSLv1 probes described below.
Negative controls
· Structural negative (3xSLv1)
· Usually highly replicated on the array; used to measure on element background. This probe forms a hairpin and does not hybridize well with labeled sample of any species
· Biological negatives (e.g., NC1_00000002)
· These are probes shown not to have significant signal from any sample tested. These probes are used to estimate on element background.
· Currently there are 82 different negative control NC probes.
· Common practice is to have one highly replicated NC probe (>100) and the other NC probes at lower replication (≈6).
· Reserve negatives (e.g., SM_01)
· There are currently 12 sequences replicated 40 times marked as control type Positive that are reserved negative controls for future potential use as positive controls. They do not show significant signal from any sample tested.
Positive controls
· Biological positives (e.g., RnPC_10045851, PC_00000004)
· Endogenous “species-specific” sequences predicted to have high signal, due to multiple copies found in the targeted genome, are used as positive controls. This is the same probe sequence used as the BrightCorner probe.
· Common practice is to have this probe highly replicated.
· Deletion stringency probes (e.g., DCP_008001.0)
· Probes designed against endogenous “species-specific” sequence can be used to measure hybridization and wash stringency.
· Approximately 20 probes, predicted to perform well by in silico metrics, are chosen randomly from a tiling database and printed on the array in triplicate. In addition, four variants of this probe are printed on the array, also in triplicate. The first variant has a one base pair deletion, the second a three base pair deletion, and the third has a five base pair deletion, and finally the fourth variant has a seven base pair deletion. Bases to be deleted are chosen at random from the center of the probe sequence. The number of bases deleted is indicated by the number after the period. For example, DCP_008001.0 and DCP_008001.3 are from the same parent sequence and the first probe has zero deletions while the second has three.
· Intensity curve probes (e.g., SRN_800001)
· Approximately two thousand species-specific probes are chosen to have predicted signals that span the practical signal space. These signal intensities are predictions made using an in-house model. These probes are generally not replicated.
· These probes can be used in a non-linear normalization.