Create library by Bait Tiling (wizard)

Parameter

Instructions/Details

Design Options

Design Job Name

Type a name that will later enable you to 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 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.) Select the desired bait length. All baits will be of exactly the length you select.

• Note:
  
  · All baits in a library must be of the same length. If you intend to combine baits from two or more sources to create a library, make sure all bates have the same length.
  
  · For all users, eArray supports SureSelect Target Enrichment libraries with a bait length of 120 nucleotides.  To enable libraries for your workgroup with bait lengths of 90, 150, and/or 170 nucleotides, contact Agilent Technical Support.

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.

• 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.

• Note: Depending on your other settings, the exact density of tiling may be somewhat less than that indicated above at the extreme 5' and 3' ends of 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:

1. Beside the Genomic Target Intervals box, click Upload.
   A dialog box appears.

2. Click Browse.
   A Choose File dialog box appears.

3. Select the desired file, then click Open.
   The name of the file appears in the previous dialog box, in File Name.

4. Click Upload File.
   eArray uploads the list of intervals, and displays them in pipe-separated format in the Genomic Target Intervals box.

Genomic Avoid 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 are regions of the genome that 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 specify 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:

1. Beside the Avoid Custom Intervals box, click Upload.
   A dialog box appears.

2. Click Browse.
   A Choose File dialog box appears.

3. Select the desired file, then click Open.
   The name of the file appears in the previous dialog box, in File Name.

4. Click Upload File.
   eArray uploads the list of intervals, and displays them in pipe-separated format in the Avoid Custom Intervals box.

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.

Length

(Read-only) The bait length of the tiled baits (in nucleotides). All baits in a library are of the same length.

• Note: For all users, eArray supports SureSelect Target Enrichment libraries with a bait length of 120 nucleotides.  To enable libraries for your workgroup with bait lengths of 90, 150, and/or 170 nucleotides, contact Agilent Technical Support.

Library Size

(Read-only) Currently, eArray supports a library size of 1 x 55K. Click Show Details to see additional information about available features and the control grid that will be applied.

Control Grid

(Read-only) 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 bait length.

This required Agilent control grid contains baits that are used for quality control purposes in the library manufacturing process.

Folder

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

Description

(Optional) Type a brief description for the library.

Species

(Read-only) Reflects the species that you selected in the previous step of the wizard.

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:

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. 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 library.

Task

Instructions/Details

Bait Group Details

Add bait
group(s)

To add bait group(s) to your library:

1. Click Add.
   A bait group selection page appears.

2. 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.

3. Do any of the following to navigate through the list of bait groups.

• To navigate among multiple pages of results, use the Pages links.

• To sort the list based on any column, click the heading of the desired column. To reverse the order of the sort, click the column heading again.

• To restrict the list to a specific folder, select the desired folder in Folder, then click Filter.

4. Click the name of the desired bait group to select it. You can select more than one bait group on a page.

5. Click Add.
   eArray transfers the selected bait group(s) to the Selected Bait Group list.

6. Do any of the following to change the Selected Bait Group list:

• Add another bait group.

• Do another bait group search. You can add bait groups from multiple searches.

• Add all of the bait groups visible on a page — Click Add All.

• Start over  — Click Remove All.

• Remove a bait group — Select the desired bait group, then click Remove.

7. Click Done. You may need to scroll down to see this button.
   The selected bait groups appear in Bait Group Details in the main window of the wizard.
   To add additional bait groups to the library, click Add again.

8. Note:
   · Use the Library Statistics pane at the bottom of the wizard to monitor the size of your library. Be sure the Percentage Filled does not exceed 100%.
   · All baits in a library must be of the same length

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

To remove a bait group from the library:

1. Mark the check box next to the name of the bait group you want to remove.

2. Click Remove.

Change control type of bait group

Next to the desired bait group, in the Control Type column, select an option from the list. The option that you select has no effect on the composition of the library. eArray attaches the designation to the bait group for user reference.

• Note: For microarray application types, control types for probe groups have these meanings:
  
  · 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.
  
  · 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).
  
  · 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.
  
  · 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.
  
  All user probe groups for the SureSelect Capture Array and microRNA application types have a control type of biological.

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).