Research

Targeted Sequencing

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 EvoPrep, KAPA EvoPlus V2, 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 EvoPrep, KAPA EvoPlus V2, 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)

KAPA HyperCap Workflow v3 Portfolio

Nucleic Acid Extraction

The Roche family of MagNA Pure Systems dramatically reduces the handling errors associated with manual techniques by automating and simplifying nucleic acid extraction.

Sample QC

Determine the probability of sequencing success for low quality samples prior to library construction using qPCR-based technology. Explore a qPCR-based NGS library quantification and quality control kit for reliable assessment of library concentration and quality in a single assay.

Library Preparation

KAPA NGS Library Preparation Kits include streamlined kits for DNA & RNA, and deliver high-quality sequencing libraries suitable for Illumina platforms.

Target Enrichment

Enrich your target regions of interest using KAPA HyperCap and KAPA HyperPETE Workflows.

Library Quantification

Quantify NGS libraries reliably and accurately with DNA standards and qPCR primers produced with stringent quality control. Amplify diverse DNA fragments with comparable efficiency and maximum lot-to-lot consistency using the KAPA Library Quantification Kits containing KAPA SYBR FAST DNA Polymerase engineered using our directed evolution technology. MC-US-01271

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

Featured Solutions for Hybridization-based Targeted Sequencing Workflow

DNA Library Preparation

Ensure high library complexity with efficient library conversion. The robust chemistries of KAPA HyperPrep and KAPA HyperPlus Kits lead to greater conversion of input DNA into adapter-ligated molecules, improving target coverage following enrichment and reducing duplicate reads.

Design Custom NGS Target Enrichment Panels with Ease

HyperDesign is a new online tool that uses Roche’s renowned algorithm for probe design and selection, enabling you to customize your target enrichment panels to meet the unique needs of your research. In addition, Roche’s Support Scientists are ready to help you with design planning and optimization.

KAPA Target Enrichment - Better by Design

KAPA Target Enrichment Probes are manufactured with KAPA HiFi DNA Polymerase and demonstrate extremely high sequence fidelity, high on-target rates, and high accuracy and reproducibility.