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

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.