【摘要】 目的 比较4种全基因组扩增试剂盒对滤纸血样本中恶性疟原虫选择性全基因组扩增的性能，为恶性疟原虫全基因组研究提供参考。方法 对恶性疟患者全血样本进行梯度稀释，制备高、中、低原虫密度的滤纸血样本并提取DNA,采用Transgen、Vazyme、QIAGEN和NEB 4种全基因组扩增试剂盒进行选择性全基因组扩增(sWGA)。扩增产物经酶切法构建测序文库，使用Illumina NovaSeq 6000 S4平台进行150 bp双端测序。测序数据经fastp进行质控，并以恶性疟原虫Pf3D7参考基因组进行比对，分析Mapping率、覆盖度和测序深度，同时评估Kelch13基因区域的扩增效果，使用R软件对数据分析并可视化。结果 4种全基因组扩增试剂盒均能满足测序文库构建需求，平均产生35.14M(30.21M～44.35M)有效测序reads,测序数据Q30比例均>85%。不同试剂盒的扩增产物在测序数据质量、Mapping率、覆盖度及测序深度方面存在显著差异，且受原虫密度影响。在低原虫密度样本中，Transgen试剂盒表现最佳，其Mapping率、覆盖度及测序深度均显著高于其他试剂盒(P<0.05)。在中等原虫密度样本中，Transgen和Vazyme试剂盒的覆盖度较高(>98%),NEB试剂盒的覆盖度波动较大(69.51(28.30)%。在高原虫密度样本中，4种试剂盒的测序数据质量差异较小，Mapping率和覆盖度均接近100%。Kelch13基因区域的测序分析显示，Transgen试剂盒在低密度样本中的测序覆盖度最高(98.39(3.23)%,NEB和QIAGEN试剂盒的覆盖度较低，且不同试剂盒在不同原虫密度下的平均测序深度差异显著。结论 不同试剂盒对恶性疟原虫选择性全基因组扩增性能的差异较大，Transgen和Vazyme对低原虫密度的样本扩增效果均较好。更多还原

【Abstract】 Objective This study evaluates four WGA kits for selective whole-genome amplification of Plasmodium falciparum from dried blood spots, providing a reference for P. falciparum whole-genome research. Methods Whole blood samples from Plasmodium falciparum-infected patients were serially diluted to prepare dried blood spot(DBS) samples with high, medium, and low parasite densities. Genomic DNA was extracted and subjected to selective whole-genome amplification(sWGA) using four different WGA kits: Transgen, Vazyme, QIAGEN,and NEB. Amplified products were used to construct sequencing libraries via enzymatic fragmentation, followed by 150 bp paired-end sequencing on the Illumina NovaSeq 6000 S4 platform. Sequencing reads were quality-controlled using fastp and aligned to the Pf3D7 reference genome. Mapping rate, genome coverage, and sequencing depth were analyzed, and the amplification performance of the Kelch13 gene region was specifically evaluated. Data analysis and visualization were performed using R. Results All four WGA kits successfully met the requirements for sequencing library construction, producing an average of 35.14 million(30.21M-44.35M) high-quality reads with Q30 scores above 85%. Significant differences were observed among kits in sequencing quality, mapping rate, genome coverage, and depth, particularly under varying parasite densities. In low-density samples, the Transgen kit performed best, with significantly higher mapping rate, coverage, and depth(P<0.05). For medium-density samples, Transgen and Vazyme achieved >98% coverage, while NEB showed large variability(69.51%(28.30%). In high-density samples, all kits yielded comparable performance with near-complete coverage. The Transgen kit also showed the highest coverage of the Kelch13 gene region in low-density samples(98.39%(3.23%),while NEB and QIAGEN performed less consistently. Conclusion The selective whole-genome amplification performance of different kits varied considerably, with Transgen and Vazyme showing better amplification efficiency in low-density Plasmodium falciparum samples.更多还原