Enrichment Panels and DesignStudio FAQ for the Illumina 5 Base WGS and Enrichment kits

What enrichment panels are compatible with this kit (off-the shelf and/or custom)? Does any enrichment panel work or does it need to be specifically designed for this kit’s chemistry? Would panels designed for bisulfite sequencing work?

• Labs must design Illumina Custom Enrichment Panel v2 panels in Illumina DesignStudio that are designed for the Illumina 5-Base DNA Prep chemistry. (eg, it is not possible to use an existing Illumina Custom v2 panel designed for Illumina DNA Prep with Enrichment in the Illumina 5-Base DNA Prep with Enrichment workflow, as it does not account for the conversion of methylated Cs).

• Additionally, there is a pre-designed Methylation Control Panel that labs can purchase from Illumina via DesignStudio to use with the GDMC and SFMC controls.

• Illumina optimizes the panel design for the unique methylation conversion chemistry specific to Illumina 5-Base DNA Prep with Enrichment. Panels designed for bisulfite sequencing will not work, as bisulfite converts un-methylated C to T, whereas Illumina 5-Base does the opposite (methylated Cs are converted to T.)

What is the process to design a custom enrichment panel for Illumina 5-Base DNA Prep with Enrichment? What are the minimum and maximum requirements (eg, probe number, target number, or region size)? Do panels use single-stranded or double stranded probes? How many bases are the probes?

• Custom panels can be designed in Illumina DesignStudio using the option for Illumina 5-Base DNA Prep with Enrichment (which is based on the Illumina Custom Enrichment Panel v2 chemistry, but specific to 5-Base Enrichment).

• Make sure that the enrichment probe panel meets the following specifications: 75 kb - 1 Mb panel size *

• Minimum probes required for ordering = 2,500 unique probes

• Maximum probes for ordering = 1,000,000 unique probes

If ordering a custom panel in DesignStudio, how many samples (and/or enrichment reactions or volume) does one order support?

• There is the option to order panels in one of three sizes: 32 µl, 384 µl, or 1536 µl.

• For each 4-plex enrichment reaction, 4 µl of panel must be used.

Can an existing Illumina Custom Enrichment Panel v2 designed for Illumina DNA Prep with Enrichment (IDPE) be used for Illumina 5-Base DNA Prep with Enrichment workflow?

• No, the probes of the IDPE panel do not account for the methylation conversions that are performed in the Illumina 5-Base DNA Prep with Enrichment, prior to probe hybridization and pulldown.

• To create a 5-Base DNA Prep with Enrichment panel from an existing Illumina DNA Prep with Enrichment panel. navigate to the IDPE panel design in DesignStudio, export all of the targets into a file, and use the file to import the targets into a new Illumina 5-Base DNA Prep with Enrichment design in DesignStudio.

• This also applies to the “Add from Existing Design” feature in DesignStudio. This option will only display probes from panels with the same assay type (ie, other Illumina 5-Base DNA Prep with Enrichment panels).

Can the kit be used with third-party panels? If so, what criteria must be met?

• Third-party panels are not supported.

Which species are supported for 5-Base panel designs?

• Only human (hg19 and hg38) and mouse (mm10, mm39) species are supported for custom Illumina 5-Base DNA Prep with Enrichment panel designs.

The purpose of the Illumina 5-Base DNA Prep with Enrichment is to interrogate methylation sites, which can vary. For a given target region, does DesignStudio make a single probe/probe set using the “typical” methylation pattern for that region (and because it is enrichment, it is still permissive to some variation) (ie, one region, one probe that accounts for mC>T conversion), or are multiple probes for the different possible methylation states designed for each target region (ie, one region, multiple probes for various mC > T conversions)?

• DesignStudio Sequencing will create one-to-many probe sets to provide coverage for a target region, depending on the size of the region. Each probe set contains 4 probes, one for each possible methylation variation.

Are 5-Base DesignStudio designs de novo? Or is DesignStudio pulling from a database of pre-designed probes?

• Designs are de novo, meaning they are calculated independently for each design and not from a database of pre-designed probes. The only caveat is if probe designs are imported using the "Add from Existing Design" feature (which requires either previous designs, or available Illumina Panels). In this case, the existing designs will be copied into the new custom panel unaltered.

What does "Tilling" mean? What is the recommendation for tiling?

Tiling meaning:

• 0.75X spacing means 160 bp on average between centers of neighboring probes (40 bp gap).

• 1X spacing means 120 bp on average between centers of neighboring probes (adjacent).

• 1.3X spacing means 120 bp on average between centers of neighboring probes (30 bp overlap).

• 2X spacing means 60 bp on average between centers of neighboring probes (60 bp overlap).

The recommendation is 1X, which is the default.

Is there a recommendation for padding?

• The recommendation for padding depends ultimately on experimental aims. Padding is a feature used to add intronic bases around CDS to the target definition to capture variants in splice sites. In the context of the Illumina 5-Base DNA Prep with Enrichment, it makes sense to use padding to study SNPs near CDS in addition to CpG sites. Conversely, it would not make sense to add padding if, for example, interested in only intronic CpG sites.

For any feedback or questions regarding this article (Illumina Knowledge Article #9944), contact Illumina Technical Support [email protected].