Create library from bait upload (wizard)

Parameter

Instructions/Details

Library Type

Select between creating a Normal library and a Supplemental library. A supplemental library is intended to be paired with an Agilent catalog library to create a combined library. Supplemental libraries can contain up to 57,750 baits, including the required Agilent quality control baits. A normal library is not paired with an Agilent catalog library, but if desired, it can later be used to create a combined library using the Combine Libraries wizard.

If you select Supplemental as the library type, you need to then make additional selections:

• Base Library Name drop-down list: Select the Agilent catalog library that you want to pair with the supplemental library when you later create the combined library.

• Ignore intervals already covered in base library check box: Mark the box if you want to skip adding baits to the supplemental library that overlap with genomic regions already covered by the chosen catalog library.

Library Category

(Read-only, SureSelect RNA Enrichment application type only) eArray supports RNA Capture libraries, which retrieve specific RNA species from a pool of RNAs.

Bait Parameter Details

Species

Select the species associated with your baits. You can use this information later to search for the baits.

Remove replicate baits from upload

A replicate bait has the same Bait ID as another bait in the file. If you mark this option, eArray uploads the first bait in each set of replicate baits in your file, and ignores the others.

• Note: If your bait file contains replicate baits, and you do not mark Remove replicate baits from upload, eArray displays an error message after you begin the upload, and does not upload your file.

Bait Precedence

These options specify what you want eArray to do if it finds baits in your uploaded file that have the same Bait ID as baits that already exist in the system. Select one of these options:

Overwrite matching baits  – The annotation of the matching uploaded bait replaces the annotation of the existing bait. This option is useful when you want to re-annotate baits.

Skip matching baits  – eArray ignores the matching uploaded baits, and only uploads new ones.

Cancel upload if any baits already exist – eArray cancels the entire upload process if an uploaded bait has the same Bait ID as a one that already exists in the system.

Bait Upload File Details

Bait Group Name

After you upload your bait data file, eArray creates a bait group that contains all the baits from the file.  Type a name for this bait group.

Upload File

To specify your bait sequence file, follow these steps. Your file must satisfy the requirements described in Preparing a file of baits for upload.

1. Click Browse.
   A dialog box appears.

2. Select the desired bait 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 Preparing a file of baits for upload.

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

• Note: If you use Microsoft Excel 2007 to create the file, save the file as an Excel 97-2003 workbook. This saves the file in the required *.xls format.

Length

Currently, eArray supports a bait length of 120 nucleotides. eArray uploads your file only if the length of every bait in your file matches this number.

Tiling Frequency

(SureSelect RNA Enrichment application type only) Select the tiling frequency that applies to the baits in your uploaded file. Use the following as a guide:

• 1x – Baits are tiled end-to-end over each target in a single layer.

• 2x – Baits overlap each other by 50% so that two baits cover each base in each target.

• 3x – Baits overlap so that three baits cover each base in each target.

• 4x – Baits overlap so that four baits cover each base in each target.
  

• 10x – Baits overlap so that ten baits cover each base in each target.

Parameter

Instructions/Details

Library 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 the library in search results, lists, and the like.

Length

(Read-only) The length of baits in your uploaded file appears here. 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) The species you selected in Step 1 of the wizard appears here.

Library Size

(Read-only) eArray supports a library size of 1 X 55K, which can accommodate up to 57,750 baits, including any required Agilent quality control baits.

For the SureSelect Target Enrichment application type, click Show Details to see additional information about available features and the control grid that will be applied. In addition, for this application type, eArray automatically creates a library set when you design a library that contains more than 57,750 baits.

Control Grid

(SureSelect Target Enrichment application type 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.

Show Other Library Details (The properties below appear if you mark Show Other Library Details.)

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 later.

Attachment

(Optional) Attachments are files or links that are related your library. Attached files are limited in size to 32 MB. 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. 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 library.

Task

Instructions/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.

1. • To go to another page of results, click one of the Pages links.

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

   • To limit 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:

4. • 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.
   The selected bait groups appear under Bait Group Name 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, click the desired link.

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. In the Select column, mark the check box next to the name of the bait group that you want to remove.

2. 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 only lets you change the Replicate values for bait groups if you select Maximize capacity in Agilent Recommended Bait Boosting. Otherwise, eArray enforces a Replicate value of 1 for all bait groups in the library.
  
  • 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 exceed the capacity of the library format (12,000 x 5 = 60,000 baits).

Change bait boosting preference

Bait boosting increases the representation of "orphan" and GC-rich baits within the library by an Agilent-defined factor. Agilent applies bait boosting to libraries by default.

More info about bait boosting

To change the bait boosting preference for the library

1. In Agilent Recommended Boosting, select one of these options:

   • Maximize Capacity – eArray applies no bait boosting to the library. This option lets you include the greatest number of user-defined baits in the library.

   • Balanced – eArray replicates any orphan or GC-rich baits present in your library by an Agilent-defined factor, potentially improving the library's capture of genomic fragments. This option uses a greater number of the available baits in the library than the Maximize Capacity option, but it uses fewer baits than the Maximize Performance option. Also, if you select this option, eArray enforces a Replicate value of 1 for all bait groups in the library.

   • Maximize Performance – To maximize the library's capture of genomic fragments, eArray replicates any orphan or GC-rich baits based on a sliding scale that reflects the boosting in Agilent’s catalog exome libraries. Baits with a higher GC content are replicated by a higher factor. This option can result in much higher levels of bait boosting than the Balanced option and, as a result, it uses the greatest number of the available baits in the library. Because the Maximize Performance option significantly boosts all GC-rich baits, Agilent does not recommend this option for libraries that cover non-coding regions with an atypically high GC content.

A dialog box asks if you are sure you want to change your boosting preference.

2. Click OK.
   Under Library Statistics, the value in Number of Available Features changes accordingly.