DNA聚合酶对PacBio SMRT测序结果影响的评价
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摘要
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.
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.
引文
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[14]Zheng Y,Xi X,Xu H,et al.Using PacBio long-read highthroughput microbial gene amplicon sequencing to evaluate infant formula safety[J].J Agric Food Chem,2016,64(37):6993.
[15]BAI Na.Diversity of intestinal microbiota of healthy pastoral livestock in Inner Mongolia XilinGol[D].Hohhot:Inner Mongolia Agricultural University,2014:1-5.(in Chinese)白娜.内蒙古锡林郭勒牧区健康家畜肠道菌群多样性研究[D].呼和浩特:内蒙古农业大学,2014:1-5.
[16]Jones MB,Highlander SK,Anderson EL,et al.Library preparation methodology can influence genomic and functional predictions in human microbiome research[J].Proc Natl Acad Sci U S A,2015,112(45):14024-14029.
[17]Thomas V,Clark J,DoréJ.Fecal microbiota analysis:an overview of sample collection methods and sequencing strategies[J].Fut Microbiol,2015,10(9):1485.
[18]Schloss PD,Gevers D,Westcott SL.Reducing the effects of PCR amplification and sequencing artifacts on 16SrRNA-based studies[J].PLoS One,2011,6(12):e27310.
[19]Biesbroek G,Sanders EAM,Roeselers G,et al.Deep sequencing analyses of low density microbial communities:working at the boundary of accurate microbiota detection[J].PLoS One,2012,7(3):e32942.
[2]HUANG Weiqiang,ZHANG Jiachao,ZHENG Yi,et al.The diversity of gut microbiota of Bai ethnic group[J].Microbiol China,2015,42(3):504-515.(in Chinese)黄卫强,张家超,郑艺,等.白族成年人肠道菌群多样性研究[J].微生物学通报,2015,42(3):504-515.
[3]Cultrone A,Tap J,Lapaque N,et al.Metagenomics of the human intestinal tract:from who is there to what is done there[J].Curr Opin in Food Sci,2015,4:64-68.
[4]Mosher JJ,Bowman B,Bernberg EL,et al.Improved performance of the PacBio SMRT technology for 16SrDNA sequencing[J].J Microbiol Methods,2014,104:59-60.
[5]Koren S,Schatz MC,Walenz BP,et al.Hybrid error correction and de novo assembly of single-molecule sequencing reads[J].Nat Biotechnol,2012,30(7):693-700.
[6]CAO Chenxia,HAN Wan,ZHANG Heping.Application of third generation sequencing technology to microbial research[J].Microbiol China,2016,43(10):2269-2276.(in Chinese)曹晨霞,韩琬,张和平.第三代测序技术在微生物研究中的应用[J].微生物学通报,2016,43(10):2269-2276.
[7]Becker S,B9ger P,Oehlmann R,et al.PCR bias in ecological analysis:a case study for quantitative Taq nuclease assays in analyses of microbial communities[J].Appl Environ Microbiol,2000,66(11):4945-4953.
[8]ZHANG Min,CAI Junpeng.Influences of different DNA polymerase for denaturing gradient gel electrophoresis(DGGE)[J].J Anhui Agric Sci,2011,39(14):8231-8233.(in Chinese)张敏,蔡俊鹏.不同DNA聚合酶对PCR-DGGE技术的影响[J].安徽农业科学,2011,39(14):8231-8233.
[9]Kim M,Oh HS,Park SC,et al.Towards a taxonomic coherence between average nucleotide identity and 16SrRNA gene sequence similarity for species demarcation of prokaryotes[J].Int J Syst Evol Microbiol,2014,64(2):346-351.
[10]Hamady M,Knight R.Microbial community profiling for human microbiome projects:Tools,techniques,and challenges[J].Genome Res,2009,19(7):1141-1152.
[11]WU Rentuya.Study on the microbial diversity of traditional fermented dairy products by 454pyrosequencing[D].Hohhot:Inner Mongolia Agricultural University,2014:13-16.(in Chinese)乌仁图雅.应用454焦磷酸测序技术对传统发酵乳制品微生物多样性的研究[D].呼和浩特:内蒙古农业大学,2014:13-16.
[12]Mosher JJ,Bernberg EL,Shevchenko O,et al.Efficacy of a3rd generation high-throughput sequencing platform for analyses of 16SrRNA genes from environmental samples[J].J Microbiol Methods,2013,95(2):175-181.
[13]J Li,H Xu,Z Sun,et al.Effect of dietary interventions on the intestinal microbiota of Mongolian hosts[J].Sci Bull,2016,61(20):1605-1614.
[14]Zheng Y,Xi X,Xu H,et al.Using PacBio long-read highthroughput microbial gene amplicon sequencing to evaluate infant formula safety[J].J Agric Food Chem,2016,64(37):6993.
[15]BAI Na.Diversity of intestinal microbiota of healthy pastoral livestock in Inner Mongolia XilinGol[D].Hohhot:Inner Mongolia Agricultural University,2014:1-5.(in Chinese)白娜.内蒙古锡林郭勒牧区健康家畜肠道菌群多样性研究[D].呼和浩特:内蒙古农业大学,2014:1-5.
[16]Jones MB,Highlander SK,Anderson EL,et al.Library preparation methodology can influence genomic and functional predictions in human microbiome research[J].Proc Natl Acad Sci U S A,2015,112(45):14024-14029.
[17]Thomas V,Clark J,DoréJ.Fecal microbiota analysis:an overview of sample collection methods and sequencing strategies[J].Fut Microbiol,2015,10(9):1485.
[18]Schloss PD,Gevers D,Westcott SL.Reducing the effects of PCR amplification and sequencing artifacts on 16SrRNA-based studies[J].PLoS One,2011,6(12):e27310.
[19]Biesbroek G,Sanders EAM,Roeselers G,et al.Deep sequencing analyses of low density microbial communities:working at the boundary of accurate microbiota detection[J].PLoS One,2012,7(3):e32942.