ARDEP, a Rapid Degenerate Primer Design Pipeline Based on <i>k</i>-mers for Amplicon Microbiome Studies

• Main Authors: Yueni Wu, Kai Feng, Ziyan Wei, Zhujun Wang, Ye Deng
• Format: Article
• Language: English
• Published: MDPI AG 2020-08-01
• Series: International Journal of Environmental Research and Public Health
• Subjects: bioinformatic program; primer design; <i>k</i>-mer; analysis platform; primer assessment
• Online Access: https://www.mdpi.com/1660-4601/17/16/5958

The survey of microbial diversity in various environments has relied upon the widespread use of well-evaluated amplification primers for taxonomic marker genes (e.g., prokaryotic 16S and fungal ITS). However, it is urgent to develop a fast and accurate bioinformatic program to design primers for microbial functional genes to explore more mechanisms in the microbial community. Here, we provide a rapid degenerate primer design pipeline (ARDEP) based on the <i>k</i>-mer algorithm, which can bypass the time-consuming step of sequence alignment to greatly reduce run times while ensuring accuracy. In addition, we developed an open-access platform for the implementation of primer design projects that could also calculate the amplification product length, GC content, Annealing Temperature (Tm), and ΔG of primer self-folding, and identify covered species and functional groups. Using this new platform, we designed primers for several functional genes in the nitrogen cycle, including <i>napA</i> and <i>amoA</i>. Our newly designed primers achieved higher coverage than the commonly used primers for all tested genes. The program and the associated platform that applied the <i>k</i>-mer algorithm could greatly enhance the design and evaluation of primers for environmental microbiome studies.