Overcoming Sequencing Challenges with WES and Custom Panels

As genomic DNA samples can often be collected only once, researchers cannot afford to repeat sequencing time and time again to achieve good coverage. For example, formalin-fixed, paraffin-embedded (FFPE) tissues— generally collected from cancer patients for histopathological diagnosis—are difficult to extract good quality DNA from due to specimen processing protocols.1 Researchers may only have one chance at obtaining good NGS results from these samples. However, certain genomic regions are notoriously difficult to sequence, which is a cause for concern when samples are limited. These difficult regions can result in non-uniform coverage of target exome regions. New tools and techniques, such as probe and panel optimization, are now available to overcome challenges associated with regions that are problematic for sequencing.

Sequencing Difficulties

During NGS library preparation, DNA molecules are fragmented, ligated to adapters suitable for the particular sequencer used, size selected, and amplified using PCR. Many enzymatic steps within library construction protocols have the potential to introduce sample composition bias. A likely source of bias is the PCR amplification step because amplification is not uniform among fragments. GC-rich or AT-rich fragments are not amplified as efficiently as other fragments, leading to notable inaccuracies in sequencing results over several rounds of amplification.

The human genome reference assembly (GRCh38/hg38 release) contains approximately 300,000 single nucleotide polymorphisms (SNPs) in regions with ≥75% GC content, leaving many potentially interesting variants difficult to access with NGS.2,3

Hybridization-based target enrichment of DNA sequencing libraries can enable sequencing of these difficult regions, especially when well-designed, high-quality probe panels are used.

Panel Selection

Whether you are performing WES, using some other predesigned panel, or using a custom probe panel, correct panel selection is critical for successfully accessing difficult genomic regions, for obtaining uniform capture, and for increasing foldenrichment of target sequences.

Several specialized hybridization probe panels are available to help researchers overcome sequencing challenges. These include probe panels that have been extensively optimized for the latest release of the human genome assembly (GRCh38/hg38) as well as panels for model organisms such as mouse (GRCm39) and zebrafish (GRCz11). Optimized panels enable researchers to increase sequencing throughput, achieve better coverage compared to standard panels, and improve uniformity across GC-rich regions.

Probe Optimization

Tools that enable researchers to create custom target enrichment designs are also now available. These take advantage of online optimization algorithms. Scientists can enter such information as gene names, sequence identifiers, and genomic coordinates, which are then processed through various algorithms to output custom-designed probe sequences.

Custom-designed and optimized probes allow researchers to improve target enrichment panels to meet unique research needs, such as for specific cancers or rare disease research. Optimized probes and panels enable scientists to perform research more efficiently and to discover variants more easily.

By using well-designed exome panels for WES, or custom panels designed to target their regions of interest, scientists can obtain proficient, robust, and automatable workflows, even for difficult-to-sequence areas of the genome. Better uniformity in sequencing means that scientists can increase sequencing depth and throughput, which also reduces the amount of resources required to uncover variants relevant to their research questions.


  1. P. Robbe et al., "Clinical whole-genome sequencing from routine formalinfixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project," Genet Med, 20:1196–1205, 2018.
  2. A. Mowjoodi et al., "Discrimination of SNPs in GC-rich regions using a modified hydrolysis probe chemistry protocol," Biotechniques, 57:313-16, 2014.
  3. Y. Guo et al., "Improvements and impacts of GRCh38 human reference on high throughput sequencing data analysis," Genomics, 109(2):83-90, 2017.

Learn more about KAPA Target Enrichment

Discover robust new products for hybridization-based target capture, with streamlined workflows that focus sequencing resources on regions of interest. Increase efficiency and depth of targeted resequencing for human genetic disease and cancer research, as well as other applications. The KAPA Target Enrichment portfolio offers solutions for:

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Learn more about KAPA Target Enrichment