NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions

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. Author summary The polymerase chain reaction is an extensively applied technique that helps to identify bacterial and viral pathogens, germline and somatic mutations. And primers are one of the most important parts of each PCR-assay, defining its sensitivity and specificity. Many tools have been developed to design primers automatically, however, none of them let someone develop multiplex PCR assays requiring to design from two to thousands of primers with one-two commands. Here we present such a tool called NGS-PrimerPlex that was initially developed to create new targeted NGS-panels. This approach has become widespread since it allows targeting only the regions of interest of a genome reducing the analysis cost. We showed that NGS-PrimerPlex can be applied for the multiplex pathogen detection and sequencing exons of the LRRK2 gene in Parkinson's disease patients. The primers designed were validated on 354 DNA samples for which the LRRK2 coding sequences were successfully studied on the MiniSeq Illumina platform.