This topic describes how to use the Create Library by Bait Tiling wizard within the SureSelect RNA Enrichment application type. A version of this wizard is also available for the SureSelect Target Enrichment application type. See Bait Tiling wizard (SureSelect Target Enrichment).
For the SureSelect RNA Enrichment application type, Bait Tiling is a process that creates baits that evenly cover specific transcript sequences or genomic regions of a given species. You can then use these baits for RNA capture, a process that isolates specific RNAs for sequencing. See SureSelect RNA enrichment libraries.
If you want to tile baits based on an uploaded file of transcript target sequences, prepare a FASTA format file that contains the desired sequences. Enter transcript sequences in 5' to 3' orientation as the coding strand of the corresponding DNA sequence. Each transcript can be up to 120 kb in length, and the total targeted sequence length can be up to 5 Mb. Use only the characters A, C, G, and T. The file must be a *.txt file in compressed (zip) format.
If you want to tile baits based on an uploaded file of GenBank accessions for specific transcripts, prepare a text file with an extension of *.txt that contains one accession 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 want to tile baits to the sequences of particular genomic intervals, you can enter them directly in the first step of the wizard, or upload them. Make sure that the intervals correspond to the currently available genome build of your species of interest in eArray. To see the name of the currently available genome build for your species, go to the first step of the wizard, then select a species. If you want to upload a file of genomic intervals, create a text file with an extension of *.txt that contains one interval on each line. Each interval can be up to 120 kb in length, and the total targeted sequence length can be up to 5 Mb. For genomic intervals, use the format shown in this example: chr17:1005000-1007650
eArray uses a specified transcriptome as a similarity database to identify potential cross-hybridization problems. Agilent recommends that you use one of the Agilent-provided transcriptomes for this purpose, if one exists for your species of interest. However, you can also upload a custom transcriptome file. If you want to use an uploaded transcriptome, create a FASTA format file that contains the desired transcript sequences. The file must be a *.txt file in compressed (zip) format.
This wizard leads you through the steps that are summarized below.
To start the wizard
Step 1 – Library Options and Target Details – In this step, you set the parameters for a Bait Tiling job, and enter and/or upload target and transcriptome data, and you submit a bait tiling job to eArray.
Step 2 – Upload Message – In this step, a message informs you that you will receive an e-mail when your design job is finished. The wizard stops here. After the Bait Tiling job finishes, you can continue.
Step 3 – Define Library – In this step, you define general attributes of the library, such as its name and location.
Step 4 – Layout Baits – In this step, you can add (or remove) bait groups, and select the number of copies of each bait group to include in the library.
Step 5 – Create Library – In this step, you save the library with a selected status. If you select a status of Submitted, eArray submits your custom library to Agilent Manufacturing, which makes it available for price quotes, and lets you purchase it online.
Click the Workspace tab. Note: Bait tiling is not available in a collaboration.
Set the application type to SureSelect RNA Enrichment. The Home tab of the SureSelect RNA Enrichment application appears.
In the Library Wizards pane, select Create Library by Bait Tiling, then click Next. The first step of the library appears in a new window.
In this step of the wizard you set the options for a Bait Tiling job.
Set the options described in the table below.
Parameter
Instructions/Details
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.
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 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.
Bait Length
(Read-only) eArray supports a bait length of 120 nucleotides.
Bait 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.
(Upload Options)
(Available if you select Transcript targets in Target Type) Select one of these options:
Upload in FASTA Format – 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.
Upload as 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:
Click Browse. A Choose file dialog box appears.
Select the desired file, then click Open. The name of the file appears in the wizard.
Click Upload File. eArray uploads the selected file.
Genome Build
(Read-only, appears if you select Genomic intervals in Target Type) Shows the genome build for the selected species that is currently available in eArray.
Genomic 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:
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.
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 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.
Click Next. eArray starts a Bait Tiling job based on the parameters that you entered.
A message tells you that you will receive an e-mail when your design job is completed.
Click Exit. At this point, the wizard pauses. An entry appears for the wizard under Search Results in the Library Wizards pane of the Home tab. You can monitor the status of the job. When the status of the job is Complete, a Continue link appears in the Actions column, and eArray sends you an e-mail. You may need to click Refresh to see the most updated version of the list of wizards, or View All to see the entry for your wizard.
In this step of the wizard, you define general attributes of the library, such as its name and location.
On your individual workspace page, in the Library Wizards pane, next to the desired wizard, click Continue. If Continue does not appear, you must wait until the Bait Tiling job finishes.
Enter the following parameters. All are required unless otherwise noted.
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.
(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:
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.
Click Next. The next step of the wizard, Layout Baits, appears.
The bait group that contains the results of the Bait Tiling job that you previously submitted in the wizard appears under Biological RNA Enrichment Bait Group Details. 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.
Use the tasks in the table below to select bait groups for your library. As you add and configure bait groups in the library, the Library Statistics pane reflects your choices. For information about these statistics, see Calculated library statistics.
Task
Bait Group Details
Add bait group(s)
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 the list of bait groups.
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.
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:
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.
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.
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:
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 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
These statistics appear:
Statistic
Number of Libraries per Slide
This value is always 1.
Number of Available Features
The number of unused features in the library to which additional baits can be assigned.
Percentage Filled
The fraction of the features in the library for which baits have been defined. This also includes the required Agilent control baits. This value must not exceed 100%.
Click Next. The next step of the wizard appears.
In this step, you save your library with a specific status.
In How do you want to save and create your Library?, select one of the following:
Draft – eArray saves the library with a status of Draft. Only you can edit the library.
Review – eArray saves the library with a status of Review. The users in your workgroup can edit the library and save versions of it.
Complete – eArray saves the library with a status of Complete. The library can be submitted to Agilent Manufacturing, and no one can edit it.
Submit – eArray saves the library with a status of Submitted, and submits the library to Agilent Manufacturing. If you select this option, you must also click Design Checklist, then read and mark all of the items that appear. Later, you can request a quote or order the library.
Click Save. eArray creates and saves your library. A success message appears.
Click Close. Another success message appears.
See also
SureSelect RNA Enrichment libraries