Multiplex PCR with the blunt hairpin primers for next generation sequencing

摘要

The multiplex PCR is one of the important methods to enrich the target DNAs for next generation sequencing. The non-specific amplification and interaction between the primers are the pivotal challenges of multiplex PCR. Here, we introduce the novel blunt hairpin primers for effective reducing the primer dimers and mispriming events. We also used a pair of auxiliary primers to enhance PCR efficiency. We simultaneously amplified 89 target regions from 44 samples and sequenced all amplicons on ion torrent PGM platform. Among all the filtrated amplicons (3438 different amplicons), 99.7, 97.6, 90.1 and 72.8% had sequencing depths fell within 200, 100, 50 and 25-fold range. The sequencing depth variations among all the samples were less than 27-fold. We also amplified multiplex regions with blunt hairpin, stick hairpin and normal linear primers, and the blunt hairpin primers could significantly reduce the amount of primer dimers and unspecific products.These results show that multiplex PCR with the blunt hairpin primers is a flexible, specific and economical target-region captured approach for the next generation sequencing.