Hybridization-Based Target Enrichment


Targeted sequencing enables researchers to enrich specific genes, exons and/or other genomic regions interest to allow sequencing reads to be dedicated to only the regions that are of interest, which results in time and cost savings.1 Various strategies are employed for enriching specific target regions, with most frequently used methods for next-generation sequencing (NGS) being hybridization or capture-based target enrichment and amplicon-based target enrichment.

How does hybridization-based target enrichment work?

Hybridization of target regions can occur either on a solid surface (microarray) or in solution.

Array-based target enrichment

In an array-based hybrid capture, genomic DNA is first sheared into randomly sized fragments. Sequencer-specific adapters are then added via a PCR reaction. An immobilized probe is used to capture the targets in the fragmented library. Nonspecific hybrids are then washed away and hybridized probes are eluted. While array-based capture methods are efficient in enriching targets of interest, they have several limitations, such as cost, limitations to the number of samples and large sample requirements.

Solution-based target enrichment

Solution-based target enrichment systems are designed to overcome the limitations imposed by array-based capture methods. A pool of biotinylated oligonucleotide probes targeting the desired genes, exons, and/or other genomic regions of interest is added to adapter-ligated DNA in solution for hybridization with targeted regions of interest.  The hybridized probes are then captured and purified by magnetic beads and subsequently amplified and sequenced.

Roche Sequencing Solutions offers SeqCap® Enrichment Kits for DNA hybrid capture-based target enrichment.

SeqCap EZ Library protocol.
1. Genomic DNA: SeqCap EZ Library oligo pool is made against target regions in the genome.
2. Library Preparation: Standard shot-gun sequencing library is made from genomic DNA.
3. Hybridization: The sequencing library is hybridized to the SeqCap EZ Library oligo pool.
4. Bead Capture: Capture beads are used to pull down the complex of capture oligos and genomic DNA fragments.
5. Washing: Unbound fragments are removed by washing.
6. Amplification: Enriched fragment pool is amplified by PCR.
7. Enrichment QC: The success of enrichment is measured by qPCR at control loci.
8. Sequencing-Ready DNA: The end product is a sequencing library enriched for target regions, ready for high-throughput next-generation sequencing.


Applications of hybrid-based target enrichment

  • Single nucleotide polymorphism (SNP) detection
  • Insertion/deletion (indel) detection
  • Variant calling


  1. Kozarewa et alCurr Protoc Mol Biol., 2015. Overview of target enrichment strategies.