Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.

多重聚合酶链反应（PCR）在分子生物学中有多种应用，包括开发新的靶向下一代测序（NGS）面板。我们介绍了NGS-PrimerPlex，这是一种高效且通用的命令行应用程序，可为不同精制类型的基于扩增子的基因组靶标富集设计引物。它支持嵌套和锚定的多重PCR，在较早构建的引物的多重反应之间重新分布以及现有NGS面板的扩展。引物设计过程考虑了二级结构的形成，池中所有引物之间的非目标扩增子，引物和频繁的基因组单核苷酸多态性（SNP）重叠。此外，NGS-PrimerPlex的用户无需手动定义输入基因组区域，因为它可以通过基因列表或外显子或密码子编号等部分自动完成。使用该程序，NGS面板可对创建了LRRK2基因编码区，并成功研究了354个DNA样品，其中至少30次读取的中位覆盖率为97.4％。为了显示NGS-PrimerPlex也可以用于细菌基因组，我们设计了引物来检测食源性病原体肠炎沙门氏菌，大肠杆菌O157：H7，单核细胞增多性李斯特菌和金黄色葡萄球菌，并考虑了基因组的可变位置。