Hybridization-Based Target Enrichment

Overview

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 That is why an integrated approach to sample preparation, for applications such as targeted sequencing, encompassing all the steps required to convert a sample to a sequencing ready library is beneficial in your research considering the precious nature of NGS samples. Therefore, we offer Roche Sample Prep Solutions, from sample collection to library quantification for different sample types and sequencing applications that are proven, complete and simple.

Various strategies can be 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. While both methods have their own advantages, solution-based target enrichment overcomes the shortcomings of array-based method, such as cost, limitations to the number of samples and large sample requirements, while retaining its advantages. This method is also easily scalable and can be easily automated.

In the solution-based method, genomic DNA is first sheared into randomly sized fragments with mechanical or enzymatic fragmentation. Sequencer-specific adapters carrying sample specific barcode sequences are then added via a ligation reaction. 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 streptavidin magnetic beads and subsequently amplified and sequenced. 

Roche Sequencing Solutions offers SeqCap® probes for DNA hybrid capture-based target enrichment.  The SeqCap probes are solution-based, designed to be specific for the target regions of interest and ready to hybridize to a sequencing library in order to enrich for targets of interest.

SeqCap EZ Workflow
1. Genomic DNA: SeqCap EZ Probes are 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 Probes.
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
  • Copy number variation (CNV) detection
  • Structural variation detection

References

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