Create baits by Bait Tiling (SureSelect RNA Enrichment)

This topic describes the Bait Tiling tool that is available within the SureSelect RNA Enrichment application type. Another topic describes the Bait Tiling tool that is available within the SureSelect Target Enrichment application type—see Bait Tiling (SureSelect Target Enrichment).

Bait Tiling creates baits that evenly cover specific transcript sequences or genomic regions of a given species. You can customize the tiling process for specific sequencing technologies and protocols, and you can also set the density of tiling. To start the Bait Tiling process, you set up a Bait Tiling job and submit it to the system. When the job finishes, you can create a bait group that contains the resultant baits. With this bait group, you can then create a SureSelect RNA Enrichment bait library that you can use for RNA capture, a process that isolates specific RNAs for sequencing. For more detailed information, see SureSelect RNA Enrichment libraries. If you like, you can use a wizard to guide you through the Bait Tiling process and the design and submission of a bait library—see the Bait Tiling wizard (SureSelect RNA Enrichment).

Note: You can use Interval Finder or Exon Interval Finder to retrieve a list of genomic intervals to use as input for the Bait Tiling process. These tools find intervals of interest based on annotations that you enter.

Before you set up a Bait Tiling job

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 text file (*.txt) in 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
If you tile baits to uploaded transcript sequences or GenBank accessions that represent them, 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 text file (*.txt) in zip format.

To set up and submit a Bait Tiling job

Click the Workspace tab. Note: Bait Tiling is not available in a collaboration for the SureSelect RNA Enrichment application type.
Set the application type to SureSelect RNA Enrichment.
Click Baits > Bait Tiling.

The Bait Tiling page appears.
Set the following parameters. All are required.

Parameter	Instructions/Details
Library Category	(Read-only) eArray supports RNA Capture libraries, which retrieve specific RNA species from a pool of RNAs.
Design Options
Design Job Name	Type a name 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 protocol(s) 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 parameter 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 may 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 text file (.txt) in 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 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. In either case, follow these steps to upload the file: Next to the desired option, 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 selected file.
Genome Build	(Read-only, appears if you select Genomic Intervals in Target Type) Shows the currently available genome build in eArray for the selected species.
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>. Example: chr21:1000000-1500000). 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. 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 location of the file appears in the appropriate box. 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 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. eArray requires a compressed (*.zip format) version of the file.

Click Submit.

eArray submits your Bait Tiling job for processing. A message tells you that you will receive an e-mail when the job is complete.
Click Close.

The Bait Tiling page reappears. Your Bait Tiling job appears in the Search Results pane at the bottom of the page, where you can monitor its status. The list of jobs does not automatically update once it appears. Click Refresh to update the list to its most current form.

Status designations and what they mean

Status	Description
In Queue	The Bait Tiling job has been submitted to the system, but no action has been taken on it yet.
Designing	The Bait Tiling process has begun.
Completed	The Bait Tiling job is finished, and the results are available for your use.
ERROR	The job was started, but there are problems. You must resubmit the job. eArray sends you an e-mail that tells you about the error. This e-mail can contain additional information about the error.

It can take up to one day or more for eArray to finish your Bait Tiling job. When eArray completes your Bait Tiling job, you receive an e-mail, and the status of the job changes to Completed. You can then view or download the results, and you can also create a new bait group from them.