Create a SureSelect RNA design

SureDesign allows you to create a custom design for SureSelect RNA target enrichment captures using a design wizard that takes you through the steps of the creation process. The wizard will ask you to define the target capture regions (using gene names or transcript IDs) and set a few of the design parameters. You then submit the design to SureDesign and the program's algorithms select the probe sequences for the design. When your probe selection job is complete, you receive an e-mail notifying you that your design is ready to be finalized. Once you finalize the design, it is available for ordering.

To open the SureSelect RNA design wizard:

·        At the top of the screen, click Create Designs > SureSelect RNA.

The wizard window opens to Step 1.

 NOTE  If you have Show Advanced Options marked, you will need to make some additional selections to open a design wizard. See Select a wizard with advanced options.

Steps of the SureSelect RNA wizard

Step 1: Define Design

In this step, complete the fields described below to define the design.

Design Name

Type a name for your design into the field. Alphanumeric characters, hyphens, underscores, and spaces are permitted. The name must be unique within your workgroup.

Species

Specify the species of the targets. The default selection is H. sapiens.

To change the selection, click Select. The Select Species dialog box opens, allowing you to select from a list of available species.

Transcriptome

Select the reference transcriptome, if available. If no drop-down list for transcriptome selection is provided, then that indicates only one database is currently available for the selected species.

Build

If multiple genome builds are available for the selected species, select the desired genome build in the provided drop-down list.

If no drop-down list for build selection is provided, that indicates that only one genome build is currently available for the selected species. That build is indicated below the Species field.

Create In

Specify the folder in which you want to save this design. The default selection is the top-level folder for your workgroup.

To change the selection, click Select to open the Select Folder dialog box, and mark the folder in which you want to save the new design. This dialog box lists the available folders within your workgroup and, if you are a member of any collaborations, lists the collaboration folders to which you have access. (If you later decide you want to change the folder location of the design, you can move it to another folder.)

Hybridization

Select the type of hybridization depending on the target enrichment reagents to be used.

·        90 Minutes - Appropriate for the 90-minute hybridization protocol used with SureSelect XT HS2 RNA reagents (including the protocol with optional overnight hold)

·        Overnight - Appropriate for the overnight hybridization protocol used with SureSelect XT RNA Direct reagents

·        Not Specified - Appropriate for designs not to be used for SureSelect target enrichment or when the SureSelect target enrichment type is still undecided

 NOTE  For information on the types of SureSelect target enrichment offered by Agilent, visit the product page at www.agilent.com/en/product/next-generation-sequencing/hybridization-based-next-generation-sequencing-ngs.

Click Next to advance to Step 2.

Step 2: Define Targets

In this step, define the target regions that you want to capture by providing the following information:

Targets

In the Targets text area, enter identifiers for the targets using either of the following approaches:

·        Type or paste the target identifiers directly into the text area. List one identifier per line.

·        Click Upload to browse to a text file (*.txt) that lists the target identifiers (one identifier per line).

The permitted identifiers are:

Gene name - enter the gene name (not case-sensitive) as it appears in one or more of the selected databases; example: brca1; see SureDesign gene finder for information on how SureDesign maps a gene name to transcripts in the reference transcriptome

Transcript ID - enter the transcript ID (not case-sensitive) as it appears in the transcriptome database selected in the previous step; examples: ENST00000357654 or NM_007294.4; if you enter the target ID without the version number, then SureDesign will identify all of the versions of the transcript present in the transcriptome

Gene ID - enter the numerical NCBI gene ID; example: 672

Allow Synonyms

When this check box is marked, SureDesign compares the gene names you entered into the Targets area to a table of synonyms, and may use the synonym names to map the genes to a location in the reference transcriptome.

In cases in which the gene name for your target is also a synonym for another gene, SureDesign treats both genes as targets when Allow Synonyms is marked. For example, if you entered DSP as a target, SureDesign would identify your target as the official gene name for desmoplakin, but it would also identify it as a synonym for the gene encoding dentin sialophosphoprotein. Consequently, the program would map the location to two completely different transcripts, and in the next step of the wizard (Step 3: Review Targets), you would see both transcript IDs listed for the target.

When the Allow Synonyms check box is cleared, SureDesign maps your targets to genomic locations using only the entered gene names.

To fully control how SureDesign maps your targets to a genomic location, enter your targets using transcript IDs or gene IDs instead of gene names.

Ignore Transcript Version (for H. sapiens, M. musculus, and R. norvegicus designs only)

Marking this check box impacts the way that SureDesign matches your inputted target identifiers to transcripts in the transcriptome. When you enter a target identifier as a transcript ID that includes the version number (e.g., ENST00000258149.10), SureDesign first searches the transcriptome for that exact transcript and version. If the Ignore Transcript Version check box is not marked, and SureDesign cannot find the exact transcript and version in the transcriptome, then SureDesign reports the target ID as not found. If, however, the check box is marked, then SureDesign repeats the search of the transcriptome, this time ignoring the version number so that any version of the transcript is included as a target.

Click Next to advance to Step 3.

Step 3: Review Targets

This step provides a chance for you to make sure that SureDesign successfully recognized all of the target identifiers that you entered in the Define Targets step. Review the Target Summary and Target Details before you proceed to the next step.

Target Summary

Near the top of the wizard window is a target summary with two bullet points that indicate:

·        1st bullet point: The number of target identifiers (target IDs) that SureDesign was able to resolve to a transcript ID within the selected transcriptome, and the total number of continuous transcript regions that comprise those targets.

·        2nd bullet point: The number of target identifiers (target IDs) that SureDesign was not able to resolve to a transcript ID, and the number of transcript IDs that are duplicates of another target identifier entered on the previous screen or that have a short sequence and cannot be used for probe selection

If SureDesign did not accurately identify all of your target regions

Target Details

The Target Details table lists the following information for each of the target identifiers that SureDesign was able to locate:

·        Target ID - The target ID is the gene name or transcript ID that you used to define the target.

·        Transcript ID - The transcript ID that SureDesign identified as a transcript of the target ID. For target IDs that were entered as gene names or gene symbols,the table lists the transcript IDs that are products of that gene. Often times, a single target ID has many transcript IDs.

·        Sequence Length - The sequence length is the total number of base pairs within the transcript ID.

Click Next to advance to Step 4.

Step 4: Enter Parameters

In this step, select the parameters for probe selection using the drop-down lists described below. You can click Reset to undo any changes you make to the parameters, and return all the parameters back to the default selection.

When you are finished making your selections, submit the design to the SureDesign job queue to begin the probe selection process.

Density

In the Density drop-down list, select the density of probes across the target regions. The options in the drop-down list range from 1x to 5x. The selected density indicates the desired number of probes per nucleotide in the target regions. For example, a density of 5x means that SureDesign tries to select probes with enough overlap for each nucleotide in the target regions to be covered by five unique probes.

The density parameter also impacts the replication of orphan probes. For a SureSelect design, a probe is considered to be an orphan if it does not overlap with other probes in the design and it is more than 100 bp away from its nearest neighbor probe on either side. A 5x-density design includes five copies of each orphan probe, a 4x-density design includes four copies of each orphan probe, and so on. However, orphan probes in a design created with a 1x density are exempt from this rule. In these designs, SureDesign duplicates all orphan probes in order to improve their capture performance to a level that is similar to that of the non-orphan probes in the design.

Boosting

When selecting probes, SureDesign can boost the number of copies of GC-rich probes in order to potentially improve the capture of genomic fragments with those probes.

In the Boosting drop-down list, select one of the options described below, or leave the default option in place.

·        No Boosting - SureDesign does not boost GC-rich probes when this option is selected.

·        Optimized Performance XT – This option is the default when the hybridization type is set to Overnight or Not Specified. This boosting algorithm is specifically optimized for the hybridization conditions in the SureSelect XT library preparation protocol to deliver optimal sequencing uniformity and read depth across the full range of GC content percentages.

·        Optimized Performance XT HS2 - This option is the default when the hybridization type is set to 90 minutes or Not specified. This boosting algorithm is specifically optimized for the hybridization conditions in the SureSelect XT HS2 library preparation protocol to deliver optimal sequencing uniformity and read depth across the full range of GC content percentages.

Filter Soft Masked

When the Filter Soft Masked check box is marked, SureDesign filters out any probes from the design that include repetitive sequences.

To submit the design for probe selection:

When you are finished setting the parameters, submit the design to the SureDesign job queue and the SureDesign algorithms will select the probes for your design.

1. Click Begin Probe Selection.
   
   A message box opens indicating the e-mail address where Agilent will contact you when the probe selection job is complete. If desired, you can enter additional e-mail addresses into the provided field.

2. Click OK in the notification message to submit the design to SureDesign.
   
   Your submission is placed in the SureDesign job queue to await probe selection.
   
   The wizard automatically advances to Step 5.

Step 5: Select Probes

At this point in the design creation process, SureDesign is processing your probe selection job. The length of time required for SureDesign to complete the job depends on the number of jobs waiting in the queue and the size of your design.

Click Close Design Wizard. When you receive an e-mail from Agilent SureDesign notifying you that your probe selection job was successfully completed, relaunch the wizard and continue creating the design:

1. Open the SureDesign Home screen.

2. Locate the design under Designs: In Progress, and click the Continue icon .
   
   The wizard window opens to Step 6.
   

 NOTE  You can monitor the status of your probe selection jobs from the SureDesign Home screen.

Step 6: Finalize/Design Complete

In this step, click Finalize Design to finish the design creation process. Alternatively, if you want to make further edits to the design, click Modify Design to delete the probes from the probe selection job and return to the Define Targets step.

 NOTE  At this step, the information in the design summary panel is complete, and you can download and view the summary files to help you decide if you want to finalize the design or revise it further.

After clicking Finalize Design, the wizard window updates to the Design Complete step, and provides the following information:

Use the action buttons at the bottom of the Finalize Design window to take further action on the design:

·        Click Order to open the Order dialog box, where you can add the design to your Agilent Genomics shopping cart or submit a request to receive a price quote.

·        Click Mark as Favorite to add the design to your list of favorites. The design will appear in the Designs: Recent and Favorites dashboard on the Home screen.

·        Click Download to download one or more design files, including a formatted PDF report that summarizes key information on the design, probes, targets, and the probe selection job.

These action buttons are also available from the design details window.