Create a microarray design/set using existing probe groups

The name of the required Agilent control grid appears as a link. Click the link to view details about it. eArray automatically selects a control grid appropriate to your design format, application type and, for some applications, species.

A control grid is a set of control probes that are added to every array design. For every Agilent microarray design format and species there is a default species specific control grid suggested in eArray. These grids contain positive controls probes designs against endogenous sequence. 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 they can be used for any species. The negative control probes are used to measure on element background. Here are some of the probe types you should 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 (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.

Parameter	Instructions/Details
Microarray Type	(ChIP application type only) Select either ChIP (chromatin immunoprecipitation array design) or CH3 (methylation array design) to designate the intended purpose of the array. eArray handles both types of designs identically, but the label follows the array through the design, manufacturing, and ordering processes.
Microarray Name	Type a name for the microarray design. eArray uses this name as one of the search keys for microarray designs, and as a way to refer to the design in search results, lists, and the like.
Design Format	Select a design format from the list. For details about the selected design format, click Show Details. When you select or change the design format, eArray recalculates the array statistics on the page. For more details about these statistics, see Calculated array statistics. You can use Array Calculator to help you select an appropriate design format. Only the design format(s) that are available for your chosen application type appear in the list.
Description	(Optional) Type a brief description for the design.
Control Grid	The name of the required Agilent control grid appears as a link. Click the link to view details about it. eArray automatically selects a control grid appropriate to your design format, application type and, for some applications, species. If appears, you can select an alternate control grid. In Control Grid, click . A list of available control grids appears in a new window. Select the desired control grid. Click Done. What is a CGH control grid and which probes does it contain? A control grid is a set of control probes that are added to every array design. For every Agilent microarray design format and species there is a default species specific control grid suggested in eArray. These grids contain positive controls probes designs against endogenous sequence. 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 they can be used for any species. The negative control probes are used to measure on element background. Here are some of the probe types you should 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 (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. Note: • For the SureSelect Capture Array application type, microarray designs do not contain any Agilent control probes. • For the Expression application type, the Agilent control grids that are available for standard Expression arrays differ from those available for Exon arrays.
Folder	Select a location for your new design. The folders to which you have access appear in the list.
Comments	(Optional) Type comments to include with the microarray design.
Feature Layout	Select one of the following: Randomized – eArray assigns probes randomly to feature locations. This is the standard feature layout. Customer Specified – This option lets you download the list of probes in the design, change the order of probes as desired, and upload the reordered list. You download the probe list after your microarray design has a status of Complete, and you upload the reordered probe list before you submit the design to Agilent Manufacturing. See How to upload ordered lists. Note: For the microRNA application type, and for CGH+SNP microarray designs, Randomized is the only option for feature layout. For the SureSelect Capture Array application type, feature layout options are not available.
Attachment	(Optional) Attachments are files or links related your microarray design. Attached files can be up to 32 Mb in size. To add one or more attachments, follow these steps: Click . A page appears in a new window. In the Attach pane, specify the following: Name – Type a name for the attachment. This name becomes the link you click to access the attachment. Type – Select the desired type of attachment from the list. An attachment can be either a file or a URL. Locale – Select the appropriate locale from the list. eArray uses this option to select content that has been localized for your region, when it is available. File – (Available only if you selected File for the type of attachment) Click Browse. Select the desired attachment in the dialog box that appears, then click Open. URL – (Available only if you selected URL for the type of attachment) Type the full URL of the Internet resource, including the protocol specifier. For example, http://www.agilent.com. Click Add. The attachment appears in the Attachment Header List at the top of the window. A success message appears. Click Close. Add additional attachments, if desired. If you add an attachment in error, select the check box next to its name in the Attachment Header List, then click Remove. Click Done. The window closes. The names of your attachments appear as links in Attachment.
Keywords	(Optional) Type new search keywords to associate with the design, separated by commas. Keywords can help you search for the microarray design later.

Task	Instructions/Details
Probe Group Details Tab
Add or remove a biological or user control probe group	You can add or remove a biological or user control probe group from your microarray design. To add a biological or user control probe group In the Probe Group Details tab, click Biological <type> Probe Group Details, then click Add. A probe group selection page appears in a new window. Select one ore more probe groups as described in Select probe groups for probe searches. To add more probe groups, click Add again. In the Control Type column, where available, select the desired control type for each probe group. See below, Change control type of probe group. To remove a biological or user control probe group In the Probe Group Details tab, click Biological CGH Probe Group Details, then mark the check box next to the probe group that you want to remove. Click Remove. Note: • Separate tasks describe how to add or remove normalization or replicate probe groups. • A CGH+SNP array must contain at least 2,000 SNP probes, and at least 5% of the total features on the array must be filled with CGH probes.
Add or remove a normalization probe group	(CGH and CGH+SNP arrays only) A normalization probe group is a special control probe group that provides data that can be used to normalize the two dye channel data generated from the array. When you create an array for certain species and design formats, eArray automatically adds a default Agilent normalization probe group. If normalization probe group(s) appear in your design, you can download a list of unique probes to use in the Agilent Feature Extraction (FE) program. The name of this file is Agilent dye normalization probe list for FE. To add a normalization probe group Click Normalization Probe Group Details, then click Add. A probe group selection page appears. Select one or more probe groups as described in Select probe groups for probe searches. The selected probe group(s) appear in Probe Group name. Note: When you add a normalization probe group to a microarray set, eArray places a copy of this probe group on every array within the set. To remove a normalization probe group Under Normalization Probe Group Details, mark the check box next to the probe group that you want to remove. Click Remove.
Add or remove a replicate probe group	(CGH, CGH+SNP, standard Expression, and Exon arrays only) A replicate probe group is a special control probe group that Feature Extraction and DNA Analytics can use to calculate the QC metric Reproducibility. These replicate probe groups are distinct from the user probe groups that you can include in designs in multiple copies. When you create an array for certain species and design formats, eArray automatically adds a default Agilent replicate probe group. To add a replicate probe group Click Replicate Probe Group Details, then click Add. A probe group selection page appears. Select one or more probe groups as described in Select probe groups for probe searches. The selected probe group(s) appear in Probe Group name. In Replicate, type the number of copies of the probe group to be included in the design. Agilent recommends a value of 5. Note: • When you add a replicate probe group to a microarray set, eArray places the specified number of copies of this probe group on every array within the set. 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. To remove a replicate probe group Click Replicate Probe Group Details, then mark the check box next to the probe group that you want to remove. Click Remove.
View a probe group	In any available Probe Group Name column, click the name of the probe group that you want to view. The View Probe Group page appears in a new window. For details about this page, see View probe group.
Change control type of probe group	(When available) Under Biological <type> Probe Group(s), next to the applicable probe group, in the Control Type column, select an option. Positive or negative user control probe groups must collectively occupy 50% or fewer of the available features in your design/set. pos – Designates the probe group as a positive user control. Although positive controls are excluded from many of the statistical QC metrics in Feature Extraction, they are available for downstream analysis. Positive controls generally have predictable signals, but this is not a requirement. An example of positive controls present on the Agilent control grid is the Agilent spike-in probes, which are used in the gene expression application for calculating QC metrics following addition of spike-in controls to the sample. neg – Designates the probe group as a negative user control. Negative control groups are intended to have no hybridization. The control grid that is assigned automatically to each design contains an adequate number of negative controls. If you assign your own additional group of negative controls, these negative controls are used by Feature Extraction for background determination (whether or not they have only background signal). biological – Designates that the probe group is not a control (condition = FALSE). It is the default choice for biological probes, which should comprise at least 50% of your design. ignore – Omits the probe group from Feature Extraction analyses and output. Once an array design is submitted, the Control Types cannot be changed, so the only way to "re-activate" them, if desired, is to modify the ControlType field of the design file. Note: • In a microarray set, if you change the control type of a probe group to either neg or pos, you must also select a probe distribution option. • For the SureSelect Capture Array and microRNA application types, the control type of all probe groups is biological.
Change number of copies of probe group	(Available for user non-control probe groups. For CGH and Expression designs, also available for replicate probe groups.) Under Biological <type> Probe Group(s), in the Replicate column, next to the desired probe group, type the number of copies of the probe group that you want to include in the microarray design. Note: For the microRNA application type, the Replicate parameter is always set to 1.
Allow eArray to create a microarray set	Mark Enable Microarray Set. If you select this option, and the number of features required to accommodate your microarray design exceeds the number of features available on one microarray slide, eArray adds as many additional slides as are needed. The Percentage Filled statistic in the third column of the Create Microarray Design pane can help you to monitor how full your microarray design is. If the percentage filled exceeds 100%, you must mark Enable Microarray Set to accommodate all of the probes of your design, or select a different design format with more feature locations. For positive or negative user control probe groups in a microarray set, you must also select a probe distribution option. Note: • Once you create an individual microarray design, you cannot subsequently convert it into a microarray set, even if you add additional probes and exceed the capacity of the selected design format. Similarly, once you create a microarray set, you cannot convert it into an individual microarray design. • Control probe distribution options are not available for the SureSelect Capture Array and microRNA application types. • You cannot create a CGH+SNP microarray set.
Change distribution of positive and negative control probe groups in a microarray set	(Available for microarray sets that have probe groups with a control type of pos or neg) eArray always puts a copy of each positive and negative user control probe group onto each microarray within a microarray set. Within each microarray in the set, the probes in the control probe groups are always assigned to random feature positions. However, you have some control over how probes are assigned to these randomly-selected feature positions from one array to another in the set. Click Biological <type> Probe Group(s). In the Control Probe Positioning column, select one of these options for each positive or negative user control probe group: Variable Random – Each positive or negative control probe appears on each microarray in the set in a different position. Example: eArray assigns Probe A randomly to feature position 4330 in one microarray in the set. In another microarray in the set, it might assign Probe A to feature position 961. Fixed Random – A given positive or negative control probe appears on each microarray in the set in the same position. Example: eArray assigns Probe A randomly to feature position 4330 in one microarray in the set. In all of the other microarrays in the set, Probe A is also assigned to feature position 4330. Note: For all other types of probe groups, eArray always assigns them to feature positions in a specific way: • Probe groups with a control type of biological or ignore – For each replicate requested, eArray distributes a single copy of the probe group randomly over all of the microarrays in the set. • Probe groups in the Replicate Probe Group Details pane or in the Normalization Probe Group Details pane – When these type(s) of probe groups are included in a microarray set, eArray places a copy of each one on every microarray in the set using Variable Random positioning. • Agilent quality control grid – With the exception of the SureSelect Capture Array application type, a set of these probes appears on every microarray in the set. The placement of these probes is the same on every array, and is defined by the control grid specifications.
Change number of features assigned to each microRNA	(microRNA application type only) In Features per microRNA, select the desired number of features from the list. This setting reflects the total number of features on the array assigned to each microRNA. Each microRNA has from one to four different probes associated with it. eArray adjusts the number of replicates of each of these probes to achieve the specified number of features per target (microRNA). A higher number generates more robust data, while a lower setting lets you measure more microRNAs per array. The default value is 16 features per microRNA target. Agilent Catalog arrays use 16 features per microRNA target for human arrays, and 20 features per microRNA target for mouse and rat arrays. You can also select values of 40 and 60 features per microRNA.
Fill unused features	You can fill the unused features of a microarray with the probe group of your choice. eArray may add only part of the probe group or more than one copy of some or all of the probe group, depending on the number of available empty features in your design, and the number of probes in the probe group. To add a whole probe group to your design, use the procedure described above in Add probe group, instead. Follow these steps to fill unused features: Mark Fill Microarrays. In Probe Group To Fill Microarray, click . A probe group selection page appears in a new window. For instructions on how to use this page, see Select probe groups for probe searches. Guidance from Agilent on unused CGH microarray features When designing a CGH microarray, if there are vacant, empty features after probes are selected, what does Agilent recommend as the next step? Should I increase the number of replicates or density? What are the pros and cons of increasing the number of replicates or increasing density? Blanks are not recommended, because Autogridding in Feature Extraction will become problematic. If the design you have selected does not fill the array, the following are Agilent recommended options, ordered by preference: 1. Choose a smaller array format. 2. Replicate all probes on the microarray. 3. Fill array using an Agilent catalog Probe Group. Also, if you prefer to increase the density of probes in the targeted genomic intervals, note that this will lead to the addition of probes that have lower scores. See FAQ “How many CGH probes and what density do I need to target a given genomic region?” (under "Creation of Probe Groups," above) for more details. Note: • For the microRNA application type, eArray always fills all empty features with a single pre-defined structural negative probe. Selection of a probe group to fill unused features is not available. • This option is not available for the SureSelect Capture Array application type. • For CGH+SNP arrays, you can fill unused features with a CGH probe group, but not a SNP probe group. • For the Expression application type, you can use an Exon probe group to fill an Exon microarray, but not a standard expression microarray.
Linker Details Tab
Add linkers to probes	Linkers are nucleic acid molecules that are added to the 3' ends of probes. They move the "active" (hybridizing) sequence farther from the glass microarray substrate. This decreases steric hindrance and makes the sequence more available for hybridization. Linker sequences themselves are designed to avoid hybridization to any sequence in the target sample. Agilent provides a default linker sequence that you can use, or you can specify your own custom linker sequence. Follow these steps to add linkers to probes: Mark Append linker to 3' end. The linker options become available. In Linker Length, select one of the following: Make probes of length – Adds nucleotides to the 3' ends of probes so that the resulting probes (active sequence plus linker) have the length specified. Type a number of nucleotides from 20 and 60 in the box. If an active probe sequence in your microarray design exceeds the length that you specify, eArray leaves it alone. It is not trimmed, and no linker is appended to it. Add linker of length – Adds the specified number of nucleotides to the 3' ends of probes. Type a number of nucleotides from 1 to 49. With this option, eArray can produce probes of up to 60 nucleotides in length. If the active probe sequences have different lengths, the resultant probes will also have different lengths after linkers are appended. If the linker sequence is shorter than the length that you specify, eArray replicates the linker sequence to fill in the length. In Linker Sequence, select one of the following: Use Agilent linker sequence – You cannot edit the sequence of the Agilent-provided linker. Use Customer linker sequence – Type a DNA base sequence for the linker. Provide a linker sequence that does not hybridize to any sequences in the target sample. For CGH microarrays, when should I choose to append linkers? For probes selected from the Agilent HD-CGH database, linkers are automatically added. For other probes, like probes generated through ‘Genomic Tiling’, Agilent recommends adding linkers in order to move the active probe sequences farther off of the array surface. This becomes more important for shorter sequences. Note: • For the SureSelect Capture Array and microRNA application types, no linker options are available. • eArray appends the Agilent linker sequence to Agilent probes, even if you select Use Customer linker sequence. • Linkers for Exon probes: Some Exon probes are designed to short exons. These probes, which are initially from 35 to 49 nucleotides in length, are padded on their 3' ends with dT nucleotides to make probes with a total length of 50 nucleotides.
Remove linkers	Clear Append linker to 3' end.