Amplification-Based Target Enrichment

Overview

Enrichment of specific genomic regions prior to sequencing offers a focused approach to next-generation sequencing (NGS) whereby sequencing reads are dedicated to regions of interest, resulting in time and cost savings.1Target enrichment can be achieved using an amplicon-based or a hybrid capture-based target enrichment strategy. In general, hybrid-based capture is preferred for large targets, such as whole exomes or large panels, while amplicon-based enrichment is desirable for smaller targeted panels. Due to primer-probe specificity being greater than hybridization of probes to target regions, higher on-target sequencing reads can be obtained using amplicon-based methods compared to hybrid-based capture. With the use of molecular inversion probes (MIPs), very high specificity can be achieved along with the ability to multiplex.

How does amplicon-based target enrichment work?

Genomic DNA is fragmented using enzymatic or mechanical methods and PCR primers or probes (such as MIPs) specific for the genes, exons and/or other genomic regions of interest are added. The fragments are amplified by PCR and then ligated to adapters or barcodes to prepare for the sequencing step. PCR could be single or multiplex generating single or multiple amplicons.

In amplicon-based target enrichment using MIPs, specific probes consisting of a common universal linker that is flanked by target-specific sequences are designed for each amplicon. The probes are then annealed to the target regions on either side, gaps between target-specific MIP sequences filled by DNA polymerase, and then ligated and circularized. Only the circularized fragments are amplified using primers specific to the common linker by PCR, while the uncircularized fragments are removed. The amplified products are then sequenced.

Roche Sequencing Solutions offers HEAT-Seq® Target Enrichment Kits, which use an advanced version of MIP technology, for amplicon-based target enrichment.

HEAT-Seq Workflow

The HEAT-Seq Enrichment System Workflow. The final product of the workflow is a sequencing-ready amplicon that contains the target DNA sequence, a molecular barcode, and sample indices. The entire workflow is completed in 8 hours with under 2 hours of hands-on time using a single tube. The protocol is easy for any lab to quickly adopt and is also automation friendly, lowering the resource burden of obtaining highly insightful biological data.


    

 

 

 

Applications of amplicon-based target enrichment

  • Single nucleotide polymorphism (SNP) detection
  • Insertion/deletion (indel) detection
  • Copy number variation detection

References

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