PCR反应条件对16S rRNA基因扩增子测序准确性的影响

Effect of PCR conditions on the accuracy of 16S rRNA gene amplicon sequencing

【背景】16S rRNA基因扩增子测序技术是一种不依赖培养而获得样本中细菌种群结构、相对丰度等信息的方法。高通量测序技术实验步骤较多,每一步骤细微的差别都可能在最终的测序结果中放大,并造成测序结果与实际情况的偏差。【目的】基于MiSeq测序平台,探讨PCR反应体系中扩增引物序列、退火温度、模板起始量、扩增循环数和变性时间等5个因素对16S rRNA基因测序结果的影响。【方法】对mock DNA的16S rRNA基因扩增子进行测序,分别分析不同的扩增引物、退火温度、模板起始量、循环数和变性时间对数据准确性的影响。【结果】不同的扩增引物对检测结果有较大的影响,采用的4组引物中,引物B(V3-V4,341F/806R)的准确性最好,引物A(V3-V4,341F/805R)次之。比较不同退火温度(52、55和60℃)对检测准确性的影响,退火温度60℃的结果最接近理论值。模板起始量(2、10和50 ng)的检测结果显示,mock DNA起始量为2ng的结果准确性最高。相较于其他3组(15+18、25+8和30+8),循环数为(20+8)的检测结果最接近mock DNA的理论值。不同变性时间(3 min和5 min)对分析结果无显著性影响。【结论】引物序列、退火温度、模板起始量和循环数是影响16S rRNA基因测序结果准确性的重要因素。

[Background]16S rRNA gene amplicon sequencing,a culture-independent approach,is commonly used to identify the bacterial community structure and relative abundance in given samples.The protocol of high-throughput sequencing technology involves many experimental steps,and the nuances of each step may be magnified in the final sequencing results,resulting in unexpected deviations from the actual situation.[Objective]With MiSeq system,we evaluated the impact of five factors in PCR on 16S rRNA gene amplicon sequencing results:primer sequence,annealing temperature,template quantity,cycle number,and denaturation time.[Methods]The 16S rRNA gene amplicon of mock community DNA was sequenced to determine the effects of these five factors on the accuracy of the results achieved.[Results]Primer had great influence on the 16S rRNA sequencing results,and primer B(V3-V4,341F/806R)showed the highest quantification accuracy among all primers,followed by primer A(V3-V4,341F/805R).Among the different annealing temperatures(52,55 and 60℃),the result of annealing at 60℃was closest to the theoretical value.As for the DNA template quantity(2,10 and 50 ng),the deviation was minimal when 2 ng DNA template was used.The result of detection with(20+8)cycles was closest to the theoretical value of mock DNA compared with that of the other three groups(15+18,25+8,and 30+8).Denaturation time(3 min and 5 min)had no significant effect on the experimental results.[Conclusion]This study revealed that primer sequence,annealing temperature,template quantity,and number of cycles were important factors affecting the accuracy of 16S rRNA gene sequencing results,which laid a foundation for the selection of standardized methods of library construction.