5 Tips for using the Nonacus Panel Design Tool

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Do you need to create a custom NGS panel?

Read our tips to find out how to create and optimise your panel design

The Nonacus Panel Design Tool

5 Tips for using the Nonacus Panel Design Tool

1. Understand the NGS terms associated with the Panel Design Tool

The Panel Design Tool uses common Next Generation Sequencing (NGS) terms, as shown in the tool interface (Figure 1A) and the panel design report (Figure 1B). To navigate quickly and efficiently around the tool, input your target regions, and understand the final panel design report, we have provided a Panel Design Tool word glossary (Table 1), which you can find at the bottom of this blog post.


Figure 1A: The Nonacus Panel Design Tool interface.


Figure 1B: The Nonacus panel design report provides design, target and probe information.

2. Select the genome build that suits your requirements

3. Decide on your regions of interest (ROIs)

4. Consider which tiling option you would like to use

The term 'tiling' refers to the number of biotinylated probes that cover each base within your target region. The tiling option you use in the design process, will influence the number of probes and impact the cost of your panel.

The Panel Design Tool offers complete user flexibility, allowing you to select the probe density that suits your NGS requirements. Under the tiling tab, you can select from either 1x or 2x tiling options. 1x tiling means that each genomic base will be covered by one probe and the probes will be aligned end-to-end. 2x tiling means that the probes are staggered across your target regions. This will create a 40-80 bp probe overlap and each base will be covered by two probes. If you would like more tiling options when using the tool, we suggest that you select the 'advanced' tiling function, which will allow you to pick from 0.05x - 20x tiling.

For NGS technologies that use end-sequencing such as Illumina, selecting 2x or higher tiling can help improve sequencing accuracy, particularly for middle regions of DNA. However, if budget is an issue, using lower tiling will decrease the number of probes in your design and decrease the price of your panel.

It is also worth considering where your design sits within our pricing tiers. It may be more cost-effective to use different tiling options to ensure your panel sits at the top of a sizing tier and maximize your panel content for the same cost per sample.

5. Consider whether you need repetitive regions in your design

Almost 50% of the human genome contains repeated DNA bases, which consist of short tandem repeats like microsatellites, as well as longer interspersed repeats such as long and short interspersed nuclear elements.3 These repeated sequences create challenges during NGS and variant detection.

To create an efficient NGS panel with excellent uniformity of coverage and on target rates, the Panel Design Tool uses integrated algorithms to automatically mask highly repetitive regions. These algorithms ensure that areas of the genome are not over-sequenced, which would waste sequencing resources, or under-sequenced which would lead to decreased sensitivity for variant detection (Figure 2).


Table 1: Glossary associated with the Nonacus NGS Panel Design Tool.

'Advanced' toggle Selecting this option within the tool will expand the tiling options within the tool.
BED file A file format type that is used to list genomic co-ordinates.
'Exome' toggle Selecting this option within the tool will automatically include validated probes from the Nonacus Whole Exome panel that are appropriate for the genes and regions specified within the tool.
Exons Region of the genome that contains the information coding for a protein.
Full gene Probes will be designed to cover the full gene including all introns, exons, and untranslated regions (UTRs).
‘Gap fill’ toggle Selecting this option within the tool will automatically include validated probes from the Nonacus Whole Exome panel in drop out/masked regions.
Gene list Gene names can be inputted into the tool in the format of one gene per line.
Genome build An accepted representation of the human genome sequence that is used by scientists and researchers as a reference.
Genomic co-ordinates This refers to a location within the gene, used to identify a genomic region of interest. Genomic co-ordinates should include both a start and end co-ordinate for the target region.
Panel design A user defined panel focused on specific regions of interest for next generation targeted sequencing.
Panel name A user defined name that is associated with each panel design that you create using the tool.
Probes Biotinylated oligonucleotide sequences that are designed to target your genomic regions of interest. The Nonacus probes are 120 bp long.
Target The region of the genome that you are interested in sequencing.
Template file A type of file format that allows the input of full genes and genomic regions to be included in the panel design. This file type can be downloaded within the tool.
Tiling Tiling refers to the number of probes that cover each base within the genome. 1x tiling: Each base will be covered by 1 probe; the probes will be designed end to end (alignment of probes). 2x tiling: Each base will be covered by 2 probes, there will be 40-80 bp overlap of the probes (overlapping probes).
Untranslated regions (UTRs) Regions of the genome that are not translated into protein, UTRs are present at the start and end of the coding regions in the mRNA strand.