Multiplex 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.

中文翻译：

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低循环数多重PCR：构建下一代测序扩增子文库的新策略

多重 PCR 是制备用于下一代测序的扩增子文库的关键步骤。然而，多重 PCR 存在一些挑战，包括均匀性差、非特异性扩增和引物二聚体。为了解决这些问题，我们提出了一种新颖的解决方案策略，该策略涉及在多重 PCR 中使用低循环数（<10 个循环），然后使用载体 DNA 和磁珠来选择目标产物。该技术提高了扩增子的均匀性，同时还减少了引物二聚体和 PCR 伪影。为了评估我们的技术，我们最初使用了来自小鼠基因组的 120 个含有单核苷酸多态性 (SNP) 位点的 DNA 片段。测序结果表明，仅经过7个循环的多重PCR，95.8%的目标SNP位点就被定位，覆盖度至少为1×。所有扩增子的平均测序深度为1705.79±1205.30×；其中 87% 的覆盖深度超过平均测序深度的 0.2 倍。与 Hi-Plex PCR (53.3%) 相比，我们的方法具有更高的均匀性 (87%)。此外，我们通过从最初的 120 个引物对中随机选择 90 个引物对两次来验证我们的策略。接下来，我们使用相同的协议来准备扩增子库。两组平均测序深度分别为1013.30±585.57×和219.10±158.27×；超过 84% 的扩增子的测序深度超过平均深度的 0.2 倍。这些结果表明，在多重 PCR 中使用低循环数是制备扩增子文库的一种经济有效的方法。