Create a microarray design from target transcripts (Wizard)

Method

Description

Base Composition Methodology

Creates probes that are all the same length, based on an empirically-determined optimal base composition profile. It is the standard GE probe design methodology. This method works best with Agilent protocols and most eukaryotic organisms.

Tm Matching Methodology

Creates probes to have similar predicted Tm. This is a good option when you design probes for prokaryotic organisms.

Parameter

Instructions/Details

Design Job Name

Type a name that will allow you to later identify this specific GE probe design job.

Probe Length

Type the maximum length for the generated probes. The allowable length is from 25 to 60 bp.

Agilent recommends a probe length of 60 bp, as this has been determined to provide the optimal balance between sensitivity and specificity for most applications on the Agilent in situ microarray platform.

Probes per target

Select from 1 to 10 probes per target from the drop-down list. This is the maximum number of probes the probe design process returns for each uploaded target sequence. The probe design process may return fewer probes than you specify.

Because of the length and high quality of the generated probes, Agilent recommends that you create one probe per target sequence. However, if you design multiple probes per target sequence, you will have the opportunity to select the best probe after a validation process.

Masking

eArray always uses both of these options in the probe design process, and they cannot be disabled.

Vector – Identifies and ignores contaminant segments during probe design. Target sequences can contain contaminant segments not actually found in the sample under study. These segments are often artifacts from cloning vectors (e.g. plasmid, phage, BAC, YAC) used in cloning and amplification processes.

Repeat – Identifies and ignores repetitive sequences within your target sequences during probe design. The genome of any given organism contains interspersed repeats and low complexity DNA sequences. These sequences, which are unique at a species level, are replicated many times throughout the genome, and are found in the transcriptome as well. Replicate regions are poor candidates for unique probes.

Probe Orientation

Select one of these options:

Sense – Produces probes in the sense or "coding strand" orientation, similar in sequence to the mRNA targets. Use this option if the sample preparation methodology yields cDNA or cRNA molecules.

Antisense – Select this option if you want probes in antisense or "template" orientation, complementary in sequence to the mRNA targets. This is the best option if your samples are directly labeled RNA. 

Design Options

Select your preferred design process. These options are only meaningful if you request more than one probe per target sequence.

Best Probe Methodology  – If there are multiple probes for each target sequence, the probe design process favors production of the highest quality probes, rather than even coverage of each target sequence.

Best Distribution Methodology – The probe design process favors even coverage of each target sequence, rather than production of the highest quality probes.

Design with 3' bias

Mark this option if you want probes derived mainly from the first 1,000 bp from the 3' end of each of your target sequences.

If you use an Agilent (or other) labeling protocol that uses linear amplification, it is important to select probes from the 3 end of the sequence. Linear amplification generates sequences that are shorter than the initial template due to the attenuation of the polymerase reaction. Because of this, most of the labeled product represents only the first 1,000 bp from the 3' end of each target sequence. It is important to design probes that represent this region.

Allow Probes to be Trimmed

(Available only for Tm-Matching methodology.) Mark this option to allow bases to be removed from candidate probes to increase compliance with the Preferred Probe Tm. eArray will not trim probes to shorter than 45 bases.

In concept, a shorter probe has less complementary sequence available, which can reduce its specificity, or infringe on its ability to form a stable duplex with the desired target. However, the risk of this occurring to a significant extent is very low.

Preferred Probe Tm

(Available only for Tm-Matching methodology.) Type the target Tm for the probe design process.

The Tm is the temperature at which equal populations of a probe and its target sequence would exist as a 50:50 mixture of duplex and single-stranded forms.  

Select a probe Tm based on two factors:

• The mean and standard deviation of the Tm of all potential probes that could be generated for the target transcriptome.

• The hybridization temperature identified in the hybridization protocol.  

In practice, the target Tm should be ~20°C higher than the hybridization temperature. For example, if the hybridization temperature is 60°C, then the target probe Tm should be 80°C.

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.

Design Format

Select a design format. For details about the selected design format, click Show Details. When you select or change the design format, an appropriate control grid appears in Control Grid.

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.

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) Reflects the species (if any) that you selected in the previous step of the wizard.

Keywords

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

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. 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. 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 check box 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.

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

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 Expression Probe Group Details, then click Add. You may need to scroll down to see this button.
   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. 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 Expression 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 a replicate probe groups.

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.

Change control type of probe group

(When available) Under 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.  

• 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.

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 number of copies of probe group

(Available for user non-control probe groups and 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.

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:

   • 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.