Guidelines for creating advanced designs

The information provided below may help you when creating a design with one of the Advanced Wizards.

Advanced methods for adding content

The advanced design wizards provide ways of adding content to a design that are not available in the standard design wizards. Agilent provides these additional methods for advanced users who want more control in their design content. These methods are summarized below.

Selecting probes from an existing design for specific genes/regions

The SureSelect and HaloPlex advanced wizards allow you to search the probes in an existing design or probegroup to find probes that cover specific genes or genomic regions of interest. You first select which existing design or probegroup will serve as the probe source. Then, you enter information on your desired targets. SureDesign locates the probes in the probe source that cover those target regions, and adds those probes to the new advanced design.

One application of this method is to create a custom version of an Agilent Catalog All Exon design that is limited to your genes or genomic regions of interest.

Selecting probes from an existing design based on Probe ID (SureSelect only) or Amplicon ID (HaloPlex only).

All probes in a design are assigned a unique identifier. For SureSelect DNA, this is called a probe ID. When using HaloPlex designs, it is called an Amplicon ID. With the advanced wizard, you can search an existing design or probegroup to find the probes with specific IDs.

This method offers a way to fine tune an existing design to include only specific probes. For example, your previous sequencing results with a particular design may reveal that certain probes in the design yield better sequencing data than others (e.g. the probes are more specific or effective at covering a region of interest). You can use the IDs to create a new version of that design that only includes the superior probes.

Selecting optimized probes (SureSelect only)

Agilent recommends the "Select Optimized Probes" option when adding content based on target genes or regions. This method uses a machine learning algorithm to select high-quality probes that cover the desired targets. Probes are selected based on multiple metrics of quality including specificity, GC content, and entropy. Probe density will vary between 1x and 5x depending on target properties such as the uniqueness of the target sequence in the genome, GC content, and target size. Notably, the algorithm ensures that the probe set does not include orphan probes (i.e., probes with no adjacent or overlapping probes).

Uploading probes (not available for HaloPlex DNA)

With the advanced wizard, you can upload your own probe sequences, creating a new probegroup containing those probe. To use this method of adding content, you provide a file containing the probe information, including sequences.

You may want to use this method for adding content if you have a set of probes that were designed with your own algorithms or were designed for a species that SureDesign does not support.

This method is also useful if you want to make edits to the probes in an existing custom or catalog design. You can download the probes from the existing design, make your edits, and then upload the edited probes.

Treatment of overlapping target regions

With any of the wizards (standard or advanced), when SureDesign selects probes based on your specified target genes or genomic regions, the program merges overlapping target regions prior to probe selection.

Because the advanced wizards allow multiple rounds of content addition, advanced designs can have multiple probegroups. Any overlapping target regions within a probegroup are merged prior to probe selection, but SureDesign does not merge overlapping target regions across probegroups. Consequently, target regions that are represented in more than one probegroup in an advanced design have proportionally more probe coverage in the design than the other target regions.

Calculation of design size

To calculate the size of an advanced design, Agilent adds together the sizes of all the individual probegroups in the design. Any overlap in the target regions between probegroups is included in the design size.

The size of an individual probegroup is the total size of its target regions of interest. Overlapping target regions within a probegroup do not contribute to the size because SureDesign merges those regions prior to selecting probes for the probegroup.