Target Enrichment

Highlights:

• Creating a new panel design for ~50 genes associated with CRC was fast and easy with the free online self-guided HyperDesign Tool—even for a user with no previous design experience.
• The design request took only ~15 minutes to enter, and the resulting panel design file was received within an hour of submission without requiring additional adjustments—enabling the panel and accessory reagents to be ordered right away.
• The resulting probe panel (ordered from Roche) yielded excellent sequencing results—confirming that the HyperDesign Tool is a fast, powerful, and accurate method for designing custom NGS panels.

Colorectal cancers are the second leading cause of cancer deaths both worldwide and in the U.S. impacting people as young as their 20’s⁽¹⁾. Thus, it is essential that oncology researchers have access to tools that will help with early identification of tumors. While a large number of CRC susceptibility genes and variants are known, many DNA sequencing workflows capture much larger regions of the genome—driving up sequencing costs, slowing down analysis, and requiring large amounts of data storage. A more efficient approach to sequencing CRC-associated regions is to focus sequencing reads on only the desired genomic regions through target-enriched NGS.

Boshen Gao M.Sc., a Roche Applications Scientist, recognized the need for a more focused CRC panel, and decided to design one using Roche’s self-guided online HyperDesign Tool. Boshen did not have any previous design experience prior to this experiment.

After logging into the online web-based interface and naming his design, Boshen uploaded a .BED file containing the genomic coordinates of 50 genes related to CRC. The target region included both coding and non-coding regions, for a total target sequence size of ~4.7 kbp. Other parameters are shown in Table 1. The entire design process took about 15 minutes. 

In less than an hour, Boshen received a confirmation email notifying him that the design process was complete. He did not have to adjust his request parameters, received no vague error messages from HyperDesign Tool, and did not need to speak directly to a design scientist. This contrasts with design tools he tested from other vendors, which either took much longer, yielded numerous unclear error messages that had to be addressed, or were not capable of designing the required panel with desired sequencing metrics.

The final target enrichment panel design provided an estimated coverage of ~4.3 kbp, or 92.09% of the desired target sequences. This was an expected result, as the target region supplied to HyperDesign tool included challenging-to-design non-coding regions, which are known to contain repetitive elements. Designs from other vendors did not yield satisfactory projected sequencing metrics. 

The confirmation email from HyperDesign Online Tool also provided a list of reagents that were needed to set up the NGS hybrid capture reactions. Therefore, Boshen was able to go ahead and order the probes and the associated reagents after receiving the HyperDesign confirmation email.

When the probe panel arrived Boshen tested it with both gDNA and cfDNA samples, following the provided Instructions for Use to set up a hybrid capture reaction using KAPA HyperCap Workflow reagents. Sequencing results from the very first experiment, using both sample types, yielded ample sequencing reads and low error rates. Subsequent analysis showed excellent fold-80 penalty, on-target rate, coverage depth, and uniformity (Table 1). The fold-80 penalty from this panel design was ~1.5, which shows great uniformity.

Taken together, this case study demonstrates that someone who needs a custom target enrichment panel but has little to no design experience can easily use the self-guided Roche HyperDesign Online Tool to design a high-quality panel, enabling significant savings of both time and lab resources.

80%+ coverage was expected due to this being a human DNA panel, with some repetitive regions that are expected to have low coverage. Panel coverage exceeded the expectation at 92%.

Table 1- Sequencing metrics for Roche CRC panel design with Roche HyperDesign Tool. Only cfDNA  data is shown here. gDNA data was comparable.

A challenging part of the project was the complexity of the design process itself–especially for the lab member running the project, who did not haveany prior experience in NGS target enrichment panel development.

The researcher in charge of the project, Boshen Gao, M.Sc. turned to the Roche HyperDesign Online Tool. This self-guided, cloud-based platform offered an intuitive workflow for designing custom target-enrichment panels. Boshen was able to quickly input the 50 target genes of interest, including challenging intronic regions known to harbor important variants. The HyperDesign Tool's pre-balancing algorithms streamlined the design process, eliminating any need for subsequent optimization

To complete the workflow, Boshen received an email within an hour that listed all of the needed probes and reagents to order, with their associated catalog numbers.  KAPA EvoPrep Library Prep Kit and the KAPA HyperCap Workflow were then used to prepare the target-enriched libraries.

For the researchers at Roche, the HyperDesign Online Tool–combined with Roche’s high-quality double stranded probes and the KAPA library preparation and target enrichment workflows–has proven to be a powerful solution for overcoming the challenges of an NGS panel design. The tool's ease of use and self-guided design, along with pre-optimized algorithms, were particularly beneficial. Thus, this tool can enable even those with limited prior panel design experience to create high-quality panels on the first try.