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

摘要:

【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2 ng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。

关键词:16S rRNA基因;反应条件;靶向测序

Abstract:

[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.

Key words:16S rRNA gene;reaction conditions;targeted next-generation sequencing