By Victoria Simms on December 20, 2022 | PANEL DESIGN TOOL | CUSTOM NGS PANELS
5 Tips for using the Nonacus Panel Design Tool
Get great coverage for the genes you care about
Do you need to create a custom NGS panel?
Read our tips to find out how to create and optimise your panel design
At Nonacus, we have developed an intuitive, online Panel Design Tool that allows you to create custom NGS panels with high on-target rates and uniform coverage - first time!
In this blog, we describe 5 tips for using the Nonacus Panel Design Tool, which will enable you to create a design within minutes.
The Nonacus Panel Design Tool
At Nonacus, we understand the importance of an effective NGS panel delivering uniform coverage and high on-target rates. That is why we have developed the Panel Design Tool.
The cloud-based tool allows you to create panels focused on the exact genomic regions that you are interested in. Using integrated algorithms, the tool ensures the probes capture target regions efficiently, enabling robust variant calling, high sample throughput and lowers your sequencing cost.
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
When creating a design, the Nonacus Panel Design Tool allows you to select from either the GRCh38 or GRCh37 genome reference builds.
All genome reference builds are updates from the initial DNA sequence released from The Human Genome Project completed in 2003.
The latest human reference genome is the Genome Research Consortium human build 38 (GRCh38) also known as Human Genome build 38 (Hg38). This build was published in 2013 and contains corrected sequencing artifacts, fewer gaps, and more alternate loci compared with the previous GRCh37 (b37/Hg19) assembly1, which was released in 20092.
If you are working on a new project, Nonacus recommend using the newest GRCh38 build. However, if your research began using the previous GRCh37 build, it is worth considering using this version of the reference genome instead.
3. Decide on your regions of interest (ROIs)
When designing your panel, you will need to determine which genomic regions to target using the probes. The Panel Design Tool has flexible file input options, so that you can choose the format that works for you.
Within the tool you can input your ROIs using:
- Browser Extensible Data (BED) files,
- gene lists or
- both (via a template file).
BED files (.bed) are tab-delimited text files, commonly used for genomic bioinformatic analysis. This file type offers a simple way to define your ROIs using genomic co-ordinates. When listing genomic co-ordinates, you will need to include the chromosome number, as well as the start and end locations. These co-ordinates will relate to the genome build that you selected in the previous step.
However, if your ROIs include full genes as well as genomic co-ordinates, we suggest using a template file, which can be downloaded from within the Panel Design Tool.
To save yourself time, it may be worth checking to see if your ROIs are already included in a Nonacus catalogue panel for oncology, prenatal healthcare, or constitutional genetics. You can use the Panel Design Tool to add to or modify one of these extensively tested and validated NGS panels.
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 1 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 2 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 maximise 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 elements3. 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).
Figure 2. A successful NGS panel needs to deliver uniform coverage of target regions so that genomic regions are not over or under-sequenced.
However, the Panel Design Tool is completely flexible for your requirements. If you would like to include the repetitive regions in your design, you can easily unmask them by using the ‘Gap fill’ option in the tool settings. By selecting ‘Gap fill’, the tool will automatically include validated probes from the Nonacus Whole Exome panel that cover those masked regions.
It is also possible to add probes from the Nonacus Whole Exome panel that are already designed to target genes and regions you have specified in your design by using the ‘Exome’ option in the tool settings.
You are now ready to start designing your custom NGS panel. However, if you would like more information on how to create a panel, you can click here to watch our how to guide video.
If you have any further questions or would like any support when using the Panel Design Tool, please contact support@nonacus.com and a member of our team will be touch.
Table 1. Word Glossary associated with the Nonacus NGS Panel Design Tool.
| WORD | DEFINITION | '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. |
References
- Genome Assembly GRCh37/hg19. Genome Reference Consortium. NCBI. NIH.Gov.
- Genome Assembly GRCh38/hg38. Genome Reference Consortium. NCBI. NIH.Gov.
- T.J Treangen & S.L Salzberg. (2011) Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet, 13 (1), pp. 36-46.