Review microarray designs/sets

Parameter

Instructions/Details

Microarray Name

Type a new name, if desired.

Design Format

The current microarray design format appears. To change it, select a new one. For details about the selected design format, click Show Details. When you change the design format, eArray recalculates the array statistics on the right side of the Edit Microarray Design pane. For more details about these statistics, see Calculated array statistics.

Only the design formats available for your chosen application type appear in the list. Some application types support only one design format.

Status

For designs/sets in review, the status can only be changed to Complete. To do this, select Complete. Only the owner of a design/set or a collaboration lead can change its status. For more information, see Overview of microarray design/set status.

Description

(Optional) Type a new brief description for the design.

Control Grid

The name of the Agilent control grid appears as a link. Click it to view details about the control grid. eArray automatically selects a control grid appropriate to your design format, application type and, for CGH and ChIP applications, species. If appears in Control Grid, you can select a different control grid.

To select a different control grid:

1. In Control Grid, click .
   A list of available control grids appears in a new window.

2. Select the desired control grid.

3. Click Done.

What is a CGH control grid and which probes does it contain?

• 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 the design/set. The folders to which you have access appear in the list.

Comments

(Optional) Type comments to include with the microarray design. These comments will appear in the Comments column on the Version History tab, and are a good way to communicate with other reviewers regarding the design.

Feature Layout

(Available only for single microarray designs, not microarray sets) Select one of the following:

Randomized – eArray assigns probes randomly to feature locations. Agilent recommends that you use randomized 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 Provide feature order.

• Note:
  • For the microRNA application type and for CGH+SNP microarrays, Randomized is the only option for feature layout.
  
  • For the SureSelect Capture Array application type, no feature layout options are available.

Attachment

(Optional) Attachments are files or links that are related your microarray design. You can add  new attachments, or edit or remove existing ones. Attached files can be up to 32 Mb in size.

To add one or more attachments

1. Click .
   A page appears in a new window.

2. 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. In the dialog box that appears, select the desired attachment, 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. Example: http://www.agilent.com.

3. Click Add.
   The attachment appears in the Attachment Header List at the top of the window. A success message appears.

4. Click Close.

5. Add additional attachments, if desired.

6. Click Done.
   The window closes. The names of your attachments appear as links in Attachment.

To edit an existing attachment

1. Click .
   A page appears in a new window.

2. Under Attachment Header List, next to the attachment that you want to edit, click Edit.
   The details of the attachment appear.

3. Change the attributes of the attachment as desired.

4. Click Save.
   A success message appears.

5. Click Close.

6. Click Done.

7. In Attachment Header List, click Done.

To remove an attachment

1. Click .
   A page appears in a new window.

2. Under Attachment Header List, mark the check box next to the attachment you want to remove.

3. Click Remove.
   A success message appears.

4. Click Close.

5. In Attachment Header List, click Done.

Keywords

(Optional) Type new search keywords to associate with the design, separated by commas. Keywords can help you search or browse 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

1. Click Biological <type> Probe Group Details, then click Add.
   A probe group selection page appears in a new window.

2. Select one ore more probe groups as described in Select probe groups for probe searches.
   To add more probe groups, click Add again.

3. 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

1. Click Biological <type> Probe Group Details, then mark the check box next to the probe group that you want to remove.

2. Click Remove.

3. 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 a 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

1. Click Normalization Probe Group Details, then click Add.
   A probe group selection page appears.

2. 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

1. Click Normalization Probe Group Details, then mark the check box next to the probe group that you want to remove.

2. 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

1. Click Replicate Probe Group Details, then click Add.
   A probe group selection page appears.

2. 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.

3. 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

1. Click Replicate Probe Group Details, then mark the check box next to the probe group that you want to remove.

2. 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 Details, 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.  

• 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.

• 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).

• 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.

• 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.
  
  • You cannot set the control type of a SNP probe group.

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.) 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.

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.

1. Click Biological <type> Probe Group(s).

2. In the Control Probe Positioning column,  select one of these options for each positive or negative user control probe group:

1. • 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 a value of 40 or 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:

1. Mark Fill Microarrays.

2. 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

• 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:

1. Mark Append linker to 3' end.
   The linker options become available.

2. In Linker Length, select one of the following:

1. • 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.

3. In Linker Sequence, select one of the following:

1. • 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?

• 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.

Version History tab

Place a different version of the design/set in review

Each time a user saves a design/set that has a status of Review, eArray saves a copy of the design/set. Each new version of the design/set has a new, higher version number. By default, eArray always sets the original version of the design/set as the current version, and uses it as the starting point for all reviews. Only the current version can be searched, browsed, or reviewed. However, if you are the owner of the design/set, you can select any version as the current one.

1. In the Version History tab, select the version of the design/set to make current.
   A dialog box appears.

2. Click OK.
   A message tells you that the design/set version has been updated.

3. Click Close.