State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China.
Mol Biol Evol. 2023 Nov 3;40(11). doi: 10.1093/molbev/msad230.
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.
扩增子捕获是一种很有前途的系统发育基因组分析目标序列捕获方法，而设计特定进化枝的核蛋白编码基因座（NPCL）扩增引物对于其成功应用至关重要。在本研究中，我们开发了一个称为 UPrimer 的引物设计程序，它可以根据基因组数据快速设计特定进化枝的 NPCL 扩增引物，而无需人工干预。与其他可用的引物设计程序不同，UPrimer 使用嵌套 PCR 策略，大大提高了设计引物的扩增成功率。我们检查了所有可获得的保存在 NCBI 中的后生动物基因组数据，并使用 UPrimer 为 21 个后生动物群开发了 NPCL 引物对，涵盖了广泛的分类群，包括节肢动物、软体动物、刺胞动物、棘皮动物和脊椎动物。平均而言，每个特定进化枝的 NPCL 引物对包含约 1000 个 NPCL。在 6 个后生动物群中进行了 PCR 扩增测试，开发的引物显示出超过 95%的 PCR 成功率。此外，我们在鳞翅目进行了一个系统发育案例研究，展示了如何使用 NPCL 引物进行基于扩增子捕获的系统发育基因组分析。我们的结果表明，使用 100 个 NPCL 探针可以恢复蝴蝶之间强有力的高级系统发育关系，突出了新设计的 NPCL 引物对在系统发育研究中的实用性。我们预计，自动化工具 UPrimer 和为 21 个后生动物群开发的 NPCL 引物对将使研究人员能够更高效、更具成本效益地获得系统发育数据，并加速生命之树各个部分的解决。
