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.

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.

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