Targeted Sequencing

Focus sequencing resources on genomic regions of interest

What is targeted sequencing?

Targeted next-generation sequencing (NGS) is a precise, powerful tool that enables in-depth genomic analysis of diseases and disorders that are driven by somatic or germIine variants, as well as analysis of many infectious diseases.

There are two principle methods for targeted sequencing: amplicon-based sequencing which relies on PCR amplification, and hybridization capture which uses complementary probes to capture target sequences. While each method has unique strengths, both enable the user to focus their sequencing efforts on genomic regions of interest, including coding and/or noncoding regions across the human genome, or in the genomes of model organisms or pathogens.

What advantages does targeted sequencing offer over WGS?

This focused approach yields deeper sequencing of target regions with fewer total reads compared to whole-genome sequencing (WGS), reducing the amount of sequencing needed and the time required for analysis. The greater depth of coverage enabled by targeted NGS increases the accuracy of sequencing data; this is especially important in variant calling in cancer research, identification of rare disease-associated mutations, and analysis of single-gene disorders.

The focused approach of targeted sequencing may also aid the development of targeted therapy applications and personalized medicine. For example, targeted resequencing of the polymorphic human leucocyte antigen (HLA) gene aids in HLA typing, which is crucial for matching in hematopoietic stem cell or solid organ transplantation. These methods can also be rapidly adapted to meet emerging health threats, such as SARS-CoV-2.

Targeted Sequencing Workflows

Hybridization-based target enrichment workflows

In most hybrid capture methods, the process starts with a standard library preparation workflow. First, input DNA is fragmented by shearing or using enzymes; this will vary depending upon the sample type and the library preparation kit being used. Then adapters specific for the sequencing platform are ligated to the DNA fragments, forming library molecules; the adapter-ligated sample is now a sequencing library. Next, the library is incubated with pools of biotinylated oligonucleotide probes, such as KAPA Target Enrichment probes, designed to target specific regions of interest within the DNA fragment library; this is the hybridization step.  Finally, streptavidin-coated magnetic beads are used to capture the biotinylated probe/target hybrids which are then washed to remove nonspecific library fragments. The result is in a sequencing-ready library that is highly enriched for the targeted DNA.

  • Leverage more than a decade of design experience to create your custom probes pools with HyperDesign, an easy-to-use online tool
  • Enrich regions of interest with custom probe panels: KAPA HyperChoice (for human designs) or KAPA HyperExplore (for non-human designs)

Streamline target enrichment with the KAPA HyperCap Workflow v3

Successful NGS target enrichment requires libraries with maximum molecular complexity and minimal bias, followed by robust, specific target enrichment. Our streamlined KAPA HyperCap Workflow v3 delivers complex libraries by combining the high conversion rate of KAPA HyperPrep or KAPA HyperPlus Kits with NGS-QC’d KAPA Target Enrichment Probes, creating a single-vendor-supported workflow.

  • Achieve greater success with low-input or poor-quality samples when you use KAPA HyperPrep and KAPA HyperPlus Library Preparation Kits
  • Multiplex up to 16 samples in the same capture, and potentially post-capture multiplex more samples in the same sequencing lane, with KAPA Unique Dual-Indexed Adapters (UDI) Primer Mixes 1-96; 97-384 will be available soon
  • Reduce workflow complexity and hands-on time with KAPA Universal Enhancing Oligos, eliminating the need for adapter-matched blocking oligos
  • Automate the entire KAPA HyperCap Workflow v3 without the need for a SpeedVac™—now with all hybridization and bead wash steps performed at the same temperature (55° C)

PCR-based target enrichment workflows

In PCR-based target enrichment methods (also known as amplicon-based), primer pairs flanking the regions of interest are used to amplify target regions; these reactions can be single- or multi-plex reactions. In some methods the sequencing adapters are included in the initial primers and added to the amplicons at this step, while other methods require a second “indexing PCR” reaction. In both cases, PCR products and final amplicon libraries are normalized and pooled prior to sequencing. While this method is efficient and easy to use, it is not ideal for targeting large genomic regions due to the cost of setting up multiple reactions, as well as the likelihood that not all regions will amplify equally, leading to uneven coverage due to PCR bias and dropouts; however, PCR bias can be minimized by the use of high-fidelity polymerases

  • Use amplicon-based NGS workflows for small target regions
  • Minimize dropouts and amplification bias in PCR-based target enrichment with KAPA HiFi DNA Polymerase

Targeted Sequencing Resources