Create library by Bait Tiling (wizard) in SureSelect RNA Enrichment

Parameter

Instructions/Details

Tiling Job Parameters

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 sequencing instrument that you will use.

Sequencing Protocol

Select the desired protocol. In the list, eArray shows the protocols that are available for your selected sequencing technology.

Library Category

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

Bait Length

(Read-only) eArray supports a bait length of 120 nucleotides.

Tiling Frequency

Select the desired tiling frequency. This setting controls the density of tiling. Agilent recommends that you use a Bait Tiling frequency of 2x.

• 1x – eArray tiles baits end-to-end over the target in a single layer.

• 2x – eArray overlaps baits by 50% as it tiles each target. Two baits cover each base in each target.

• 3x – eArray overlaps baits as it tiles each target so that three baits cover each base in each target.

• 4x – eArray overlaps baits as it tiles each target so that four baits cover each base in each target.
  

• 10x – eArray overlaps baits as it tiles each target so that ten baits cover each base in each target.

• Note: The exact density of tiling can differ slightly from that indicated above at the extreme 5' and 3' ends of each target.

Keep only unique baits

By default, if the Bait Tiling process produces two or more baits that have the same sequence, eArray includes only one copy of the bait when you create a bait group from the Bait Tiling results. To include all copies of such baits, clear this option.

Target Details

Target Type

Select one of these options:

• Transcript targets – Tiles baits either to the transcript sequences in an uploaded file, or to the sequences that are associated with uploaded GenBank accessions. If you select this option, the items described below in Upload Options and Transcriptome Details appear.

• Genomic intervals – Tiles baits to specific genomic intervals in a species that you enter or upload. If you select this option, the items described below in Genome Build, and Genomic Intervals appear.

Species

Select the species that is associated with the target sequences.

Content Type

(Available if you select Transcript targets in Target Type) Select one of these options:

• FASTA file – Lets you upload a FASTA format file that contains the base sequences of the transcripts to be tiled. The file must be a *.txt file in compressed (zip) format. Each transcript can be up to 120 kb in length, and the total targeted sequence length can be up to 5 Mb.

• GenBank Accessions – Lets you upload a *.txt file contains GenBank accession values, one per line. The transcripts associated with these accessions can each be up to 120 kb in length, and the total targeted sequence length can be up to 5 Mb.

In either case, follow these steps to upload the file:

1. Click Browse.
   A File Upload dialog box appears.

2. Select the desired file, then click Open.
   The name of the file appears in the wizard and will be uploaded when you click Submit to submit the tiling job.

Transcriptome

(Available if you select Transcript targets in Target Type) Select one of these options:

• Agilent – Choose this option to use one of Agilent's pre-defined transcriptomes that can be used for the probe design process. These transcriptomes, which serve as similarity databases, were constructed specifically for probe design.
  
  If you choose Agilent, you then need to select the target species from the adjacent drop-down list.
  

• GenBank Accessions – Choose this option to upload a *.txt file that contains GenBank accession values, one per line. The transcripts associated with these accessions can each be up to 120 kb in length, and the total targeted sequence length can be up to 5 Mb.
  
  If you choose GenBank Accessions, follow these steps to upload the file:

1. Click Browse.
   A File Upload dialog box appears.

2. Select the desired file, then click Open.
   The name of the file appears in the wizard and will be uploaded when you click Submit to submit the tiling job.

Learn more

(Link, appears if you select Genomic intervals in Target Type) Shows the genome build for the selected species that is currently available in eArray.

Enter Intervals

(Available if you select Genomic intervals in Target Type) Type the desired target genomic intervals in the box. Use the format chrX:<start>-<end>. Separate multiple intervals with pipe "|" characters. Each interval can be up to 120 kb in length, and the total targeted sequence length can be up to 5 Mb.

Example: chr21:1000000-1500000|chr3:1000-25000
 

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. Next to the Genomic Target Intervals box, click Upload.
   A dialog box appears.

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

3. Select the desired file, then click Open.
   The name of the file appears in the wizard and will be uploaded when you click Submit to submit the tiling job.

Transcriptome Details

Transcriptome Options

(Available if you select Transcript targets in Target Type) eArray uses the transcriptome file as a similarity database to identify potential cross-hybridization problems.

Select one of these options:

• Select Agilent-Provided Transcriptome (by Species) – Uses one of Agilent's available species transcriptome databases. If a transcriptome is available for your species of interest, select this option. These databases have been specifically constructed for use in bait design. Select the desired species from the list.

• Upload Transcriptome File – Uses a FASTA format transcriptome file that you upload as the similarity database. If you select this option, click Browse. Select the desired transcriptome file, then click Open. The location of the file appears in the box. The file must be a *.txt file in compressed (zip) format.

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 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. 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) eArray supports a library size of 1 x 55K.

Show Other Library Details (These parameters 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.

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 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:

1. • 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 Biological RNA Enrichment 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 Bait Library Statistics pane at the bottom of the wizard to monitor the size of your library. Be sure the Percentage Filled by Selected Bait Groups does not exceed 100%.
   
   • All baits in a library must be of the same length
   
   • Agilent also adds a required control bait group to your library. This control bait group is replicated the same number of times as the least-replicated bait group in your library. These control baits count against the available features in the library.

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 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 exceed the capacity of the library format (12,000 x 5 = 60,000 baits).

View library statistics

Library statistics appear under Bait Library Statistics.

More info about library statistics