Target Enrichment

Enriching target regions of interest for next-generation sequencing

Next-generation sequencing (NGS), with its ability to process hundreds of millions of DNA templates in parallel, has increased sequencing throughput and diminished sequencing costs tremendously.¹ However, whole genome sequencing is still expensive and does not provide the precision and depth of information required to decipher the roles of individual genes in complex diseases or afford the ability to investigate rare and low-frequency genetic variants. A more cost-effective way is to focus on specific regions of interest using target enrichment strategies. Target enrichment allows focusing of sequencing resources, which leads to reduced cost and simplified analysis.²

Several strategies are employed for enriching for specific genomic regions of interest before sequencing. The methods used predominantly are amplicon-based enrichment using molecular inversion probes (MIP) and hybrid capture enrichment, using probes that hybridize specifically with targeted regions of interest in solution or on a solid surface.

Roche Sequencing offers target enrichment workflows and high-quality kits for either of these methods. These workflows enable focusing sequencing resources for targeted resequencing applications in human genetic disease and cancer research.

Hyperbridization-Based Target Enrichment

Explore the KAPA Target Enrichment portfolio, which use a solution-based capture method for target enrichment. Learn about the HyperCap Workflow v3.0 that combines all reagents and accessories required for library preparation and target enrichment in a single workflow. MC-US-06356

Design Custom NGS Target Enrichment Panels with Ease

HyperDesign is a free online tool that enables you to quickly and easily design custom probes.

Primer Extension-Based Target Enrichment

Combine the performance of hybrid-capture target enrichment with the speed and simplicity of amplicon workflows with KAPA HyperPETE (Primer Extension Target Enrichment). MC-US-10049

Target Enrichment Selector Tool

Explore the Target Enrichment Selection Guide.

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Thinking of automating library prep on your existing liquid handler?

Roche’s NGS Automation Support Team, in collaboration with non-Roche liquid handling vendors, creates menus of automated methods for KAPA library prep reagents. Let’s talk about the next steps to get you up and running with automating your NGS library prep protocols. Visit support for non-Roche liquid handlers to learn more.

Support for automating KAPA reagents on non-Roche liquid handlers

Support for SeqCap Target Enrichment

Our SeqCap Target Enrichment portfolio has been discontinued. If you are an existing SeqCap user and need support, please contact our Scientific Support team for all your inquiries.

US Customers only:

Email: [email protected]
Phone: 800.262.4911

(Monday - Friday, 9 am - 5 pm EST)

References

Can Dijk et al. Trends in Genetics, 2014. Ten years of next generation sequencing technology.
Kozarewa et al. Curr Protoc Mol Biol., 2015. Overview of target enrichment strategies.

Videos

KAPA Target Enrichment

Building on over 10 years of design expertise, Roche is pleased to introduce the KAPA Target Enrichment Portfolio. These probes for hybridization-based target enrichment are available in the KAPA HyperExome or Custom Panels that you develop using HyperDesign, our online development tool.

Ask a Scientist: Low-quality samples in NGS Target Enrichment

How can high-quality NGS results be achieved with low-quality DNA samples? This video describes the challenges of working with low-quality, low-input DNA samples from a variety of sources. It also provides tips on how to increase success in next-generation sequencing (NGS) library preparation and hybridization-based target enrichment workflows when working with these samples. Presented by Dustin Masser of Roche Sequencing and Life Science.

Introduction to KAPA NGS Target Enrichment

KAPA NGS target enrichment, methods, performance metrics, and application data.

Webinar: Target-enriched RNA-seq for SARS-CoV-2 surveillance and genome analysis

New SARS-CoV-2 strains are continuing to emerge, potentially impacting the effectiveness of existing vaccines. Thus, the ability to determine the genomic sequences in samples is critical for effective surveillance and tracking of the virus. Because viral RNA represents only a small fraction of the nucleic acids in a given sample, the development of robust, sensitive sequencing methods is essential. We have developed a method for target-enriched RNA-seq with hybridization-based target enrichment (TE) of the SARS-CoV-2 genome, enabling high-throughput analysis to discover new viral mutations and track strain transmission. The workflow utilizes the KAPA SARS-CoV-2 TE panel and the HyperCap v3 workflow. In this webinar we:

Review the HyperCap v3 workflow using the KAPA SARS-CoV-2 TE panel
Describe an accelerated workflow with a shortened 1 hour hybridization
Discuss optional workflow modifications to increase SARS-CoV-2 reads
Present performance data for samples containing varying amounts of SARS-CoV-2 RNA in a background of human RNA
Show that the KAPA SARS-CoV-2 TE panel can detect mutations from multiple strains within a single reaction

Presented by Sarah Trusiak, PhD, Sr. Application Scientist at Roche Sequencing & Life Science

KAPA products are for Research Use Only. Not available for diagnostic procedures.
KAPA is a trademark of Roche.

A success story: Fast and easy design of a custom target enrichment panel for colorectal cancer (CRC) using the self-guided HyperDesign Tool

Boshen Gao, M.Sc., Application Scientist

“ I was impressed by how the HyperDesign Tool was able to help me create a robust probe panel design without a need for optimization, and that the subsequent sequencing results confirmed the expected performance of the panel.”

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.

Goal of the panel design

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.

The design process and experience

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.

Step by step instructions for the HyperDesign Online Tool design process:

Log into HyperDesign.com and follow the prompts
Submit your design for probe panel creation
Once your panel results are returned (typically in ~1 hour), review the coverage results across your target regions and approve in HyperDesign.com
Once your receive the confirmation email, order the probes and the required reagents; these are both provided in the confirmation email, enabling you to get started right away

Receiving the design files

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.

Panel performance

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.

Sequencing result:

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%.

View Full Table

Metrics
On-target rate	78-83%
Median coverage depth	78-99X
>30X coverage	70-91%
Fold-80 penalty	1.4-1.5

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

In summary, a novel colorectal cancer target enrichment panel was generated using Roche’s HyperDesign Tool, and achieved excellent estimated coverage without modification to default design parameters on the first trial. The panel design outperformed all other panel designs that were tried out from other vendors.

Chen J. et al. Cause of death among patients with colorectal cancer: a population-based study in the United States. Aging (Albany NY). 2020 Nov 28;12(22):22927-22948.

For Research Use Only. Not for use in diagnostic procedures.

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