UPrimer: A Clade-Specific Primer Design Program Based on Nested-PCR Strategy and Its Applications in Amplicon Capture Phylogenomics

Amplicon capture is a promising target sequence capture approach for phylogenomic analyses, and the design of clade-specific nuclear protein-coding locus (NPCL) amplification primers is crucial for its successful application. In this study, we developed a primer design program called UPrimer that can quickly design clade-specific NPCL amplification primers based on genome data, without requiring manual intervention. Unlike other available primer design programs, UPrimer uses a nested-PCR strategy that greatly improves the amplification success rate of the designed primers. We examined all available metazoan genome data deposited in NCBI and developed NPCL primer sets for 21 metazoan groups with UPrimer, covering a wide range of taxa, including arthropods, mollusks, cnidarians, echinoderms, and vertebrates. On average, each clade-specific NPCL primer set comprises ∼1,000 NPCLs. PCR amplification tests were performed in 6 metazoan groups, and the developed primers showed a PCR success rate exceeding 95%. Furthermore, we demonstrated a phylogenetic case study in Lepidoptera, showing how NPCL primers can be used for phylogenomic analyses with amplicon capture. Our results indicated that using 100 NPCL probes recovered robust high-level phylogenetic relationships among butterflies, highlighting the utility of the newly designed NPCL primer sets for phylogenetic studies. We anticipate that the automated tool UPrimer and the developed NPCL primer sets for 21 metazoan groups will enable researchers to obtain phylogenomic data more efficiently and cost-effectively and accelerate the resolution of various parts of the Tree of Life.