You can upload probes to eArray and create a microarray design from
the uploaded probes. This topic explains how to use the Create
a Microarray Design by Uploading Probes wizard to accomplish this.
This wizard is available for the Expression, CGH, ChIP, and SureSelect
Capture Array application types.
In this step of the wizard, you specify details about how eArray handles
the probes in your uploaded file, as well as details about the probe data
file itself.
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.
Click
Browse.
A dialog box appears.
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
|
In this step of the wizard, you identify the contents of each column
of your probe data file. The first few rows of data from your probe data
file appear under Define Uploaded File Columns.
If the first row of your probe file
is actually a row of column headings, mark My
uploaded file contains column headings. This prevents eArray from
treating the column headings in the file as a set of probe data. eArray
does not interpret any column headings in your uploaded file.
In the lists at the bottom of each column,
select the label that best matches the data in the specific preview column
above it.
If you want the upload process to ignore a specific column, select
Ignore. Use each label exactly
once, except Ignore, which you can use any number of times.
Click Upload.
eArray processes your uploaded file. A message informs you that the
file has been successfully submitted to the upload queue.
Click Close.
At this point, the wizard pauses, and you must wait for eArray to complete
the upload process before you continue. The probe upload job appears on
your workspace home page in the Pending Jobs pane. The wizard itself appears
in the Search Results section of the Create Design Wizards pane with a
status of Upload Pending.
Click Close.
It can take several days for eArray to upload the probes from your
file to your workspace, depending upon the number of users, the size of
your uploaded file, and the number of other users' jobs ahead of yours
in the queue. When the process is complete, you receive an e-mail from
Agilent. The e-mail also indicates if there was an error during the upload
process.
After eArray completes the probe upload process, the status of your
wizard in the Design Wizards pane on your workspace home page changes
to Probes Uploaded. A Continue link appears beside it in the Action column.
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:
In Control Grid, click .
A list of available control grids appears in a new window.
Select
the desired control grid, then click Done.
What is a CGH control grid and which probes does it contain?
|
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:
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.
|
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?
|
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.
|
In this step, you specify details about the probe groups in your microarray
design. You can also add additional probe groups, enable the creation
of a microarray set, and fill empty features in the microarray with the
probe group of your choice. At the bottom of the window, calculated
statistics about your microarray design appear in the Microarray Statistics
pane.
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
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 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
Click Biological <type> Probe Group Details,
then mark the check box next to the probe group that you want to remove.
Click
Remove.
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
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.
To remove a normalization
probe group
Click Normalization Probe Group Details, then
mark the check box next to the probe group that you want to remove.
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
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.
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.
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.
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.
|
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:
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
|
In this step, you save your design with a specific status.