Low cycle number multiplex PCR: A novel strategy for the construction of amplicon libraries for next‐generation sequencing

Abstract

AbstractMultiplex PCR is a critical step when preparing amplicon library for next‐generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer–dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer–dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79 ± 1205.30×; 87% of them reached a coverage depth that exceeded 0.2‐fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi‐Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer‐pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30 ± 585.57× and 219.10 ± 158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2‐fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost‐effective and efficient approach for the preparation of amplicon libraries.