A Tool to Automatically Design Multiplex PCR Primer Pairs for Specific Targets Using Diverse Templates

Overview

Journal Sci Rep

Specialty Science

Date 2023 Sep 30

PMID 37777580

Authors

Lin Yang

Feng Ding

Qiang Lin

Junhua Xie

Wei Fan

Fangyin Dai

Peng Cui

Wanfei Liu

Affiliations

Soon will be listed here.

Abstract

Multiplex PCR is an increasingly popular method for identifying species, investigating environmental diversity, and conducting phylogenetic analysis. The complexity and increasing availability of diverse templates necessitate a highly automated approach to design degenerate primer pairs for specific targets with multiple sequences. Existing tools for degenerate primer design suffer from poor maintenance, semi-automation, low adaptability, and low tolerance for gaps. We developed PMPrimer, a Python-based tool for automated design and evaluation of multiplex PCR primer pairs for specific targets using diverse templates. PMPrimer automatically designs optimal multiplex PCR primer pairs using a statistical-based template filter; performs multiple sequence alignment, conserved region identification, and primer design; and evaluates the primers based on template coverage, taxon specificity, and target specificity. PMPrimer identifies conserved regions using Shannon's entropy method, tolerates gaps using a haplotype-based method, and evaluates multiplex PCR primer pairs based on template coverage and taxon specificity. We tested PMPrimer using datasets with diverse levels of conservation, sizes, and applications, including tuf genes of Staphylococci, hsp65 genes of Mycobacteriaceae, and 16S ribosomal RNA genes of Archaea. PMPrimer showed outstanding performance compared with existing tools and experimental validated primers. PMPrimer is available as a Python package at https://github.com/AGIScuipeng/PMPrimer .

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