In this step of the wizard, you set the bait tiling options.
Parameter |
Instructions/Details |
Library
Type (Select one of these options) |
Normal |
This option creates a regular, stand-alone bait library.
Normal libraries automatically become library
sets if you include more baits than can be accommodated by an individual
library. Normal libraries cannot be included in combined
libraries. |
Supplemental |
This option creates a specialized supplemental
library that can be added to a specific Agilent Catalog library
(the "base library") when you create
a combined library. This lets you add coverage for additional regions
that are not covered in the base library. Supplemental libraries can contain
up to 57,750 baits, including the required Agilent quality control baits.
Library set
support is not available for supplemental libraries.
If you select this type of library, these additional
parameters appear:
Base
Library Name –
(Required) Select the desired base library. eArray sets certain Bait Tiling
parameters for the supplemental library to those that were used to create
the baits in the selected base library. Later, when you create a combined
library that includes the selected base library and the supplemental library,
the baits will be compatible.
Ignore
intervals already covered in base library –
(Optional) This option excludes from the genomic target intervals any
intervals that are already covered in the selected base library. This
helps to avoid the presence of duplicate baits when you create a combined
library.
|
Job Info |
Job Name |
Type a name that will later let you identify this
Bait Tiling job. |
Sequencing Technology |
From the list, select the type of DNA sequencer that
you will use to analyze your target DNA fragments. |
Sequencing Protocol |
Select the desired protocol. In the list, eArray
displays the available protocols for your chosen DNA sequencing technology.
|
Design Options |
Design Strategy |
In general, mark Use
Optimized Parameters. With this option, based on your selected
sequencing technology and protocol, eArray automatically sets the best
values for Design Strategy, Bait Length, Bait Tiling Frequency, and Allowed
Overlap into Avoided Regions.
To set all of these options manually, clear Use Optimized Parameters. The options
all become available.
In Design Strategy,
select one of these options:
Centered –
eArray centers sets of tiled baits over their respective target intervals.
Each set of baits hangs over both ends of its target interval by equal
amounts. This option exactly respects the value that you select for Bait
Tiling frequency.
Justified –
eArray designs sets of tiled baits to exactly cover their respective target
intervals. This option adjusts the overlap of baits to achieve the precise
and even tiling of only the target intervals, without any overhang into
non-target regions. With this option, the actual tiling frequency can
deviate from the value you select in Bait Tiling Frequency.
|
Bait Length |
(Available only if you clear Use
Optimized Parameters.) Currently, eArray supports a bait length
of 120 nucleotides.
|
Bait Tiling Frequency |
(Available only if you clear Use
Optimized Parameters.) Select the desired tiling frequency. This
setting controls the density of tiling. The density can also be affected
by your choice of Design Strategy. Not all bait tiling frequencies are
available for every sequencing technology.
1x
–
eArray tiles baits end-to-end. All bases in each interval are covered
by one bait.
2x
– eArray overlaps baits
by 50% as it tiles each interval. Two baits cover each base in each interval.
3x
– eArray overlaps baits
as it tiles each interval so that three baits cover each base in each
interval.
4x
– eArray overlaps baits
as it tiles each interval so that four baits cover each base in each interval.
5x
– eArray overlaps baits
as it tiles each interval so that five baits cover each base in each interval.
|
Allowed Overlap into Avoided
regions |
(Available only if you clear Use
Optimized Parameters.) To change the value, select it, then type
the desired distance in base pairs. eArray generates baits that may extend
by this distance into the regions specified in Genomic Avoid Intervals
(see below). For best results, set this distance to a maximum of 20 bp. |
Strand |
Select one of these options:
Sense –
Creates baits in sense orientation. eArray selects this option by default.
Antisense –
Creates baits in antisense orientation.
Both –
Creates both sense and antisense baits.
|
Target Details |
Species |
Select the desired species. This list contains the
names of all species currently supported in eArray for Bait Tiling. |
Genome Build |
(Read-only) This box shows the genome build of your
selected species that eArray will use to generate baits. |
Genomic Target Intervals |
Type target intervals using the format chrX:<start>-<end>
(for example, chr21:1000000-1500000). Separate
multiple intervals with pipe "|" characters.
Alternatively, you can upload a list of intervals.
Create the list as a *.txt file with one interval per line. End each line
with a new line character —
that is, in a word processor, press Enter
at the end of each line. Then follow these steps:
Next to the Genomic Target Intervals
box, click Upload.
A dialog box appears.
Click Browse.
A Choose File dialog box appears.
Select the desired file, then click
Open.
The name of the file appears in the previous dialog box, in File Name.
Click Upload
File.
eArray uploads the list of intervals, and displays them in pipe-separated
format in the Genomic Target Intervals box.
|
Extend Interval Boundaries |
If you mark this option, eArray tiles baits into
the regions that flank your entered genomic target intervals. In 5' bp and 3'
bp, type the additional number of base pairs to be added to the
5' and 3' ends of each genomic target interval. |
Avoided Genomic Intervals |
Mark any of these options:
Avoid
Standard Repeat Masked Regions –
eArray excludes a standard set of repetitive genomic regions from the
Bait Tiling process. These regions of the genome generally produce poor
quality baits. This option is marked by default.
Avoid
Custom Intervals –
In the Bait Tiling process, eArray excludes the genomic intervals that
you enter in the box. Type intervals using the format chrX:<start>-<end>
(for example, chr21:1000000-1500000). Separate
multiple intervals with pipe "|" characters. Alternatively,
you can upload a list of intervals. Create the list as a *.txt file with
one interval per line. End each line with a new line character — that is, in a word processor,
press Enter at the end of each
line. Then follow these steps:
Next to the Avoid Custom Intervals
box, click Upload.
A dialog box appears.
Click Browse.
A Choose File dialog box appears.
Select the desired file, then click
Open.
The name of the file appears in the previous dialog box, in File Name.
Click Upload
File.
eArray uploads the list of intervals, and displays them in pipe-separated
format in the Avoid Custom Intervals box.
|
A message tells you that you will receive an e-mail when your design
job is completed.
In this step of the wizard, you define general attributes of the library,
such as its name and location.
Parameter |
Instructions/Details |
Library Name |
Type a name for the library. eArray uses this name
as one of the search keys for libraries, and as a way to refer to it in
search results, lists, and the like. |
Base Library Name |
(Read-only, supplemental libraries only) The name
of the Agilent Catalog library (the "base library") that was
selected for the bait tiling job. The new supplemental library can only
be included with this specific Agilent Catalog library when you create
a combined
library. |
Length |
(Read-only) The bait length of the tiled baits (in
nucleotides). All baits in a library are of the same length. Currently,
eArray supports a bait length of 120 nucleotides. |
Folder |
Select a location for your new library. The folders
to which you have access appear in the list. |
Species |
(Read-only) Reflects the species that you selected
in the previous step of the wizard. |
Library Size |
(Read-only) Currently, eArray supports a library
size of 1 x 55K, which can accommodate up to 57,750 baits, including the
required Agilent quality control baits. Click Show
Details to see additional information about available features
and the control grid that will be applied. If you design a library that
contains more than 57,750 baits, eArray creates a library
set in which the requested baits are distributed over multiple
libraries. See library
sets. |
Control Grid
|
(Read-only) eArray automatically selects an appropriate
control grid for the library. The name of the Agilent control grid appears
as a link. To view details about the control grid, click the link.
To select an alternate control grid, if one is available,
click . A window opens where you can select the desired alternate control
grid.
Agilent has two control grids: Hs and Generic. If
you select H. sapiens as the
species, eArray automatically selects the Hs control grid. Otherwise eArray
selects the Generic control grid. These small groups do not affect your
capture and are for Agilent QC purposes. For the bait groups that you
add, generally users designate all of them with a control type of biological. If you have a control group
planned (and you want to, for example, hybridize spiked-in DNA to those
baits), then you could designate the group with a control type of Positive. This designation does not
have any special effect on the synthesis of the library. Control baits
are not listed in the BED file. |
Description |
(Optional) Type a brief description for the library. |
Keywords |
(Optional) Type search keywords to associate with
the library, separated by commas. Keywords can help you search for the
library. |
Attachment |
(Optional) Attachments are files or links that are
related your library. 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. 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 library. |
The bait group that contains the results of the Bait Tiling job that
you previously submitted in the wizard appears under Biological SureSelect
Target Enrichment Bait Group(s. In this step of the wizard, you can set
the number of copies of this bait group to include in your library, and
you can also add additional bait groups.
Task |
Instructions/Details |
Add bait
group(s) |
(Available for Normal libraries, not Supplemental
libraries) To add bait group(s) to your library:
Click
Add.
A bait group selection page appears.
In Bait Group Name, type all or part of
the name of a bait group, then click Search.
To return a list of all available bait groups, leave Bait Group Name empty.
A list of bait groups appears.
Do any
of the following to navigate through the list of bait groups.
Click the
name of the desired bait group to select it. You can select more than
one bait group on a page.
Click Add.
eArray transfers the selected bait group(s) to the Selected Bait Group
list.
Do any
of the following to change the Selected Bait Group list:
Click
Done. You may need to scroll down
to see this button.
The selected bait groups appear under Biological SureSelect Target
Enrichment Bait Group(s) in the main window of the wizard. To add additional
bait groups to the library, click Add
again.
|
View a bait group |
In the Bait Group
Name column, the names of bait groups in the library appear as
links. Click the desired link to view details about the bait group.
The View Bait Group page appears in a new window.
For details about this page, see View bait
group. |
Remove bait group |
(Available for Normal libraries, not Supplemental
libraries) To remove a bait group from the library:
Mark the
check box next to the name of the bait group you want to remove.
Click
Remove.
|
Change number of copies
of bait group |
In the Replicate
column, next to the desired bait group, type the number of copies of the
bait group that you want to include in the library.
Note:
·
eArray replicates the required Agilent control bait group the same number
of times as your least-replicated bait group.
·
eArray automatically includes as many complete sets of baits as will fit
in the available space in the library. Example:
Your library contains 12,000 baits, including the required Agilent control
baits. The capacity of the 1 X 55K library format is 57,750 baits. eArray
includes four complete sets of baits in your library (12,000 x 4 = 48,000
baits). Five complete sets would have exceeded the capacity of the library
format (12,000 x 5 = 60,000 baits).
|