If you have existing probe groups, and you want to create a microarray
design or set with them, use the method described in this topic. You can
also use this method to create microarrays with the new probe groups that
you generate in eArray. You first use eArray's Probe Group Search or Browse
commands to find and select one or more probe groups to include in your
microarray design or set. You then define design parameters and create
the design.
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?
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. |
The parameters on the page that are not described
above are either calculated by eArray or are informational only, and cannot
be edited. Of particular note are the calculated values on the page, which
track microarray statistics as you select design options and add or remove
probe groups. See Calculated
Array Statistics for a description of these values.
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.
|
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
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?
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 nt in length, are padded
on their 3' ends with dT nucleotides to make probes with a total length
of 50 nt.
|
Remove linkers |
|