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.