DNA聚合酶对PacBio SMRT测序结果影响的评价

Influence of different DNA polymerases on the results of PacBio SMRT sequencing

目的评估不同DNA聚合酶是否会对以16SrRNA全长为测序靶点的肠道微生物多样性研究结果产生影响。方法用美国太平洋公司的三代测序仪(PacBio single molecule real-time sequencing technology)对3份分别采用KAPA HiFi^(TM) HotStart DNA聚合酶和PCRBIO HotStart DNA聚合酶扩增的军犬粪便样品进行精确至"种"水平的测序分析。结果经配对Mann-Whitney U检验显示,不同DNA聚合酶扩增的同一样品在门、属和种水平上差异无统计学意义(P>0.05),然而在某些相对含量较少的操作分类单元(OTU)上,其扩增效率存在差异。经基于非加权UniFrac距离的非加权组平均法聚类分析和基于加权UniFrac距离的非参数多元方差分析发现不同DNA聚合酶扩增的同一样品其多样性差异无统计学意义(P>0.05)。结论 KAPA HiFi^(TM) HotStart DNA聚合酶和PCRBIO HotStart DNA聚合酶虽对模板DNA扩增存在一定的偏好性,但该偏好性不影响PacBio SMRT测序结果。

Objective To evaluate whether different DNA polymerases have an impact on the results of research on gut microbial diversity on the basis of full-length 16S rRNA gene as the target of sequencing.Methods Bacterial 16S rRNA gene sequences were amplified from the genomic DNA of three canine fecal samples with KAPA HiFi^TM HotStart DNA polymerase and PCRBIO HotStart DNA polymerase,respectively.The full-length 16S rRNA genes were sequenced using the PacBio single molecule real-time sequencing technology to profile gut microbial communities at the species level.Results Based on t test in a pairwise manner,the results showed there were no significant differences in the levels of phylum,genus and species between the two different DNA polymerases（P〈0.05）,but in amplification efficiency when it came to some of relatively less operating groups（OTUs）.UPGMA cluster analysis of unweighted UniFrac distance and PERMANOVA analysis of weighted UniFrac distance all showed that there was no significant difference in the diversity of the same sample amplified by different DNA polymerases.Conclusion Although both KAPA HiFi^TM HotStart DNA polymerase and PCRBIO HotStart DNA polymerase show certain PCR amplification bias,they do not affect the results of PacBio SMRT sequencing.