Create a microarray design by uploading probes (Wizard)

Parameter

Instructions/Details

Probe Parameter Details

Species

Select the species that is associated with your probes. eArray uses the selected species for several purposes:

• eArray selects the appropriate species-specific Agilent control grid for your microarray design. For Expression type designs, the species is not required, since the Agilent control grids for that application type are not species-specific.

• eArray uses species as a search key for microarray searches.

• For CGH and Expression designs and sets, eArray adds an Agilent replicate probe group, if one is available for your species and design format. Feature Extraction and DNA Analytics use data from these probes to calculate the Reproducibility QC metric.

• For CGH designs and sets, eArray adds an Agilent normalization probe group to your design or set, if one is available for your species and design format. In downstream analysis, use the data from the probes in the normalization probe group, only, to normalize the two dye channel data generated from the microarray.

Remove replicate probes from upload

A replicate probe has the same Probe ID as another probe in the file. If you mark this option, eArray uploads the first probe in each set of replicate probes in your file, and ignores the others.

• Note: If your probe file contains replicate probes, and you do not mark Remove replicate probes from upload, eArray displays an error message after you begin the upload, and does not upload your file.

Probe Precedence

These options specify what you want eArray to do if it finds probes in your uploaded file that match (have the same Probe ID and sequence as) probes that already exist in the system. Select one of these options:

Overwrite matching probes  – The annotation of the matching uploaded probe replaces the annotation of the existing probe. This option is useful for reannotating probes.

Skip matching probes – eArray ignores the matching uploaded probes, and only uploads new ones.

Cancel upload if any probes already exist – eArray cancels the entire upload process if it finds a matching uploaded probe.

Upload Probe File Details

Probe Group Name

After you upload your probe data file, eArray creates a probe group that contains all the probes from the file.  Type a name for this probe group.

Upload File

To specify your probe sequence file, follow these steps. Your file must satisfy the requirements described in Probe file formats and requirements for uploading.

1. Click Browse.
   A dialog box appears.

2. Select the desired probe data file, then click Open.
   The location of the selected file appears in Upload File.

File Format

Select either MINIMAL or COMPLETE. For details about these file formats, see Probe file formats and requirements for uploading.

File Type

Select the appropriate file type from the list. eArray accepts Microsoft Excel (*.xls) files, and tab-delimited text (TDT)  files with file extensions of .txt and .tdt

• Note: If you use Microsoft Excel 2007 to create the file, save the file as an Excel 97-2003 workbook. This saves the file in the required *.xls format.

Parameter

Instructions/Details

Define Design

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.

Microarray Type

(ChIP application type only) Select either ChIP (chromatin immunoprecipitation microarray design) or CH3 (methylation microarray 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.

Design Format

Select the desired design format. To view details about the design format, click Show Details.

When you select a design format, a control grid appropriate to the design format and application type (and, for CGH and ChIP applications, species) appears in Control Grid.

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

Control Grid

The name of the control grid appears as a link. Click the link 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 choose an alternate control grid.

To choose an alternate control grid:

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

2. Select the desired control grid, then 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.

Folder

Select a location for your new microarray design. The folders to which you have access appear in the list.

Description

(Optional) Type a brief description for the design.

Species

(Read-only) The species you set in Step 1 of the wizard appears here for informational purposes. You cannot edit it.

Keywords

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

Attachment

(Optional) Attachments are files or links that are related your microarray design. Attached files should not exceed 32 Mb in size. To add one or more attachments,  follow these steps:

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... 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.

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.
   If you add an attachment in error, select the check box next to its name in the Attachment Header List, then click Remove.

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

Comments

(Optional) Type comments to include with the microarray design.

Linker Details

Append linker to 3' end

Mark this option to add linker sequences to your 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.

If you mark this option, also set the Linker Length and Linker Sequence parameters.

For CGH microarrays, when should I choose to append linkers?

• Note: For the SureSelect Capture Array application type, no linker options are available.

Linker Length

(Available if you select Append linker to 3' end)

Select one of these options:

• 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.

Linker Sequence

(Available if you select Append linker to 3' end)

Select one of these options:

• 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.

• Note: eArray appends the Agilent linker sequence to Agilent probes, even if you select Use Customer linker sequence.

Task

Instructions/Details

Select Probes and Layout Options

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 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 below describe how to add or remove normalization or replicate probe groups.

Add or remove a normalization probe group

(CGH 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

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 and Expression 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.

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(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.  

• 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.

• 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.

• 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.

• 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 application type, 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, this is 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.

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 Microarray Statistics pane can help you to monitor how full your microarray is. If the percentage filled exceeds 100%, you must mark Enable Microarray Set to accommodate all the probes of your design, or choose 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 SureSelect Capture Array type arrays.

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.

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 a user control or non-control 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: This option is not available for the SureSelect Capture Array application type.