扩增区域对鲊广椒细菌MiSeq测序的影响
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摘要
在使用引物27F/338R、338F/806R和515F/907R分别对细菌16S rRNA基因的V_1～V_2区、V_3～V_4区和V_4～V_5区进行扩增的基础上,采用MiSeq高通量测序评价扩增区域对鲊广椒细菌多样性分析结果的影响。结果发现,扩增16S rRNA基因V_4～V_5区的非特异性序列所占比例显著偏低(P<0.05),而扩增V_1～V_2区样品细菌的Chao 1指数显著偏高(P<0.05)。虽然扩增16S rRNA基因的V_3～V_4区会导致Cyanobacteria(蓝细菌门)和Pediococcus(小球菌)相对含量显著偏高(P<0.05),但不同扩增区域扩增出的同一样品其微生物群落结构无显著差异(P>0.05)。在对鲊广椒细菌多样性进行解析时,建议选择引物515F/907R扩增16S rRNA基因的V_4～V_5区。
This study aimed to evaluate whether different amplified regions of 16 S rRNA gene influence the results of bacterial diversity in Zhaguagnjiao by MiSeq sequencing. The V_1–V_2, V_3–V_4 and V_4–V_5 regions were amplified using bacterial genomic DNA extracted from Zhaguangjiao samples as template with three different primer pairs, 27 F/338 R,338 F/806 R and 515 F/907 R. The results indicated that the percentage of non-specific amplification sequences were signi?cantly lower in the ampli?ed products of V_4–V_5 region(P < 0.05), and the Chao 1 index was signi?cantly higher in the ampli?ed products of V_1–V-2 regions(P < 0.05). Although the relative abundance of Cyanobacteria and Pediococcus were signi?cantly higher in the ampli?ed products of V_3–V_4 region(P < 0.05), there was no signi?cant difference in the microbial community structure of the same samples with different ampli?ed variable regions(P > 0.05). Thus, ampli?cation of the V_4–V_5 region using primer pair 515 F/907 R is recommended for analysis of the bacterial diversity of Zhaguangjiao.
This study aimed to evaluate whether different amplified regions of 16 S rRNA gene influence the results of bacterial diversity in Zhaguagnjiao by MiSeq sequencing. The V_1–V_2, V_3–V_4 and V_4–V_5 regions were amplified using bacterial genomic DNA extracted from Zhaguangjiao samples as template with three different primer pairs, 27 F/338 R,338 F/806 R and 515 F/907 R. The results indicated that the percentage of non-specific amplification sequences were signi?cantly lower in the ampli?ed products of V_4–V_5 region(P < 0.05), and the Chao 1 index was signi?cantly higher in the ampli?ed products of V_1–V-2 regions(P < 0.05). Although the relative abundance of Cyanobacteria and Pediococcus were signi?cantly higher in the ampli?ed products of V_3–V_4 region(P < 0.05), there was no signi?cant difference in the microbial community structure of the same samples with different ampli?ed variable regions(P > 0.05). Thus, ampli?cation of the V_4–V_5 region using primer pair 515 F/907 R is recommended for analysis of the bacterial diversity of Zhaguangjiao.
引文
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[18]BOKULICH N A,THORNGATE J H,RICHARDSON P M,et al.Microbial biogeography of wine grapes is conditioned by cultivar,vintage,and climate[J].Proceedings of the National Academy of Sciences,2014,111(1):139-148.DOI:10.1073/pnas.1317377110.
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[20]ALEGRíAá,SZCZESNY P,MAYO B,et al.Biodiversity in Oscypek,a traditional Polish cheese,determined by culturedependent and-independent approaches[J].Applied and Environmental Microbiology,2012,78(6):1890-1898.DOI:10.1128/AEM.06081-11.
[21]SAKAMOTO N,TANAKA S,SONOMOTO K,et al.16S rRNApyrosequencing-based investigation of the bacterial community in nukadoko,a pickling bed of fermented rice bran[J].International Journal of Food Microbiology,2011,144(3):352-359.DOI:10.1016/j.ijfoodmicro.2010.10.017.
[22]王玉荣,孙永坤,代凯文,等.基于单分子实时测序技术的3个当阳广椒样品细菌多样性研究[J].食品工业科技,2018,39(2):108-112.DOI:10.13386/j.issn1002-0306.2018.02.021.
[23]RAVEL J,GAJER P,ABDO Z,et al.Vaginal microbiome of reproductive-age women[J].Proceedings of the National Academy of Sciences,2011,108(S1):4680-4687.DOI:10.1073/pnas.1002611107.
[24]XU N,TAN G,WANG H,et al.Effect of biochar additions to soil on nitrogen leaching,microbial biomass and bacterial community structure[J].European Journal of Soil Biology,2016,74(3):1-8.DOI:10.1016/j.ejsobi.2016.02.004.
[25]YUSOFF M Z M,HU A,FENG C,et al.Influence of pretreated activated sludge for electricity generation in microbial fuel cell application[J].Bioresource Technology,2013,145(11):90-96.DOI:10.1016/j.biortech.2013.03.003.
[26]CAPORASO J G,KUCZYNSKI J,STOMBAUGH J,et al.QIIMEallows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.DOI:10.1038/nmeth.f.303.
[27]CAPORASO J G,BITTINGER K,BUSHMAN F D,et al.PyNAST:a flexible tool for aligning sequences to a template alignment[J].Bioinformatics,2010,26(2):266-267.DOI:10.1093/bioinformatics/btp636.
[28]EDGAR R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.DOI:10.1093/bioinformatics/btq461.
[29]HAAS B J,GEVERS D,EARL A M,et al.Chimeric 16S rRNAsequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons[J].Genome Research,2011,21(3):494-504.DOI:10.1101/gr.112730.110.
[30]COLE J R,CHAI B,FARRIS R J,et al.The ribosomal database project(RDP-II):introducing myRDP space and quality controlled public data[J].Nucleic Acids Research,2007,35(1):169-172.DOI:10.1093/nar/gkl889.
[31]DESANTIS T Z,HUGENHOLTZ P,LARSEN N,et al.Greengenes,a chimera-checked 16S rRNA gene database and workbench compatible with ARB[J].Applied and Environmental Microbiology,2006,72(7):5069-5072.DOI:10.1128/AEM.03006-05.
[32]PRICE M N,DEHAL P S,ARKIN A P.Fasttree:computing large minimum evolution trees with profiles instead of a distance matrix[J].Molecular Biology and Evolution,2009,26(7):1641-1650.DOI:10.1093/molbev/msp077.
[33]R?YTI?H,MOKKALA K,VAHLBERG T,et al.Dietary intake of fat and fibre according to reference values relates to higher gut microbiota richness in overweight pregnant women[J].British Journal of Nutrition,2017,118(5):343-352.DOI:10.1017/S0007114517002100.
[34]LOZUPONE C,KNIGHT R.UniFrac:a new phylogenetic method for comparing microbial communities[J].Applied and Environmental Microbiology,2005,71(12):8228-8235.DOI:10.1128/AEM.71.12.8228-8235.2005.
[35]ZHANG J,GUO Z,XUE Z,et al.A phylo-functional core of gut microbiota in healthy young Chinese cohorts across lifestyles,geography and ethnicities[J].The ISME Journal,2015,9(2):1979-1990.DOI:10.1038/ismej.2015.11.
[36]ZHANG J,WANG L,GUO Z,et al.454 Pyrosequencing reveals changes in the faecal microbiota of adults consuming Lactobacillus casei Zhang[J].FEMS Microbiology Ecology,2014,88(3):612-622.DOI:10.1111/1574-6941.12328.
[37]ZHANG J,GUO Z,LIM A A Q,et al.Mongolians core gut microbiota and its correlation with seasonal dietary changes[J].Scientific Reports,2014,4:5001.DOI:10.1038/srep05001.
[38]GUO Z,ZHANG J,WANG Z,et al.Intestinal microbiota distinguish gout patients from healthy humans[J].Scientific Reports,2016,6:20602.DOI:10.1038/srep20602.
[39]周书楠,席修璞,董藴,等.琚湾酸浆面浆水细菌多样性评价[J].中国酿造,2018,37(1):49-53.DOI:10.11882/j.issn.0254-5071.2018.01.011.
[40]郭壮,沈馨,董蕴,等.襄阳大头菜腌制液膜醭细菌群落结构研究[J].中国酿造,2017,36(7):143-147.DOI:10.11882/j.issn.0254-5071.2017.07.031.
[41]SOGIN M L,MORRISON H G,HUBER J A,et al.Microbial diversity in the deep sea and the underexplored“rare biosphere”[J].Proceedings of the National Academy of Sciences,2006,103(32):12115-12120.DOI:10.1073/pnas.0605127103.
[42]YOUSSEF N,SHEIK C S,KRUMHOLZ L R,et al.Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNAgene-based environmental surveys[J].Applied and Environmental Microbiology,2009,75(16):5227-5236.DOI:10.1128/AEM.00592-09.
[43]MOSHER J J,BOWMAN B,BERNBERG E L,et al.Improved performance of the PacBio SMRT technology for 16S rDNAsequencing[J].Journal of Microbiological Methods,2014,104(11):59-60.DOI:10.1016/j.mimet.2014.06.012.
[2]ILLEGHEMS K,WECKX S,DE VUYST L.Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample[J].Food Microbiology,2015,50(12):54-63.DOI:10.1016/j.fm.2015.03.005.
[3]CLAESSON M J,WANG Q,O’SULLIVAN O,et al.Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNAgene regions[J].Nucleic Acids Research,2010,38(22):e200.DOI:10.1093/nar/gkq873.
[4]吴燕燕,钱茜茜,李来好,等.基于Illumina MiSeq技术分析腌干鱼加工过程中微生物群落多样性[J].食品科学,2017,38(12):1-8.DOI:10.7506/spkx1002-6630-201712001.
[5]LIU M,TANG Y,ZHAO K,et al.Determination of the fungal community of pit mud in fermentation cellars for Chinese strong-flavor liquor,using DGGE and Illumina MiSeq sequencing[J].Food Research International,2017,91(1):80-87.DOI:10.1016/j.foodres.2016.11.037.
[6]FU J,LV H,CHEN F.Diversity and variation of bacterial community revealed by MiSeq sequencing in Chinese dark teas[J].PLoS ONE,2016,11(9):e0162719.DOI:10.1371/journal.pone.0162719.
[7]智楠楠,宗凯,杨捷琳,等.Illumina Miseq平台深度测定酸奶中微生物多样性[J].食品工业科技,2016,37(24):78-82.DOI:10.13386/j.issn1002-0306.2016.24.007.
[8]赵仁亮,胥伟,吴丹,等.基于Illumina MiSeq技术分析不同地域加工的茯砖茶中微生物群落多样性[J].生态学杂志,2017,36(7):1865-1876.DOI:10.13292/j.1000-4890.201707.012.
[9]NELSON M C,MORRISON H G,BENJAMINO J,et al.Analysis,optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys[J].PLoS ONE,2014,9(4):e94249.DOI:10.1371/journal.pone.0094249.
[10]王彦杰,徐海燕,侯强川,等.DNA聚合酶对PacBio SMRT测序结果影响的评价[J].中国微生态学杂志,2018,30(1):93-99.DOI:10.13381/j.cnki.cjm.201801024.
[11]VINCENT T,JAMES C,JO?L D.Fecal microbiota analysis:an overview of sample collection methods and sequencing strategies[J].Future Microbiology,2016,10(9):1485-1504.DOI:10.2217/fmb.15.87.
[12]SUN D L,JIANG X,WU Q L,et al.Intragenomic heterogeneity of16S rRNA genes causes overestimation of prokaryotic diversity[J].Applied and Environmental Microbiology,2013,79(19):5962-5969.DOI:10.1128/AEM.01282-13.
[13]米其利,李雪梅,管莹,等.高通量测序在食品微生物生态学研究中的应用[J].食品科学,2016,37(23):302-308.DOI:10.7506/spkx1002-6630-201623049.
[14]NAM Y D,PARK S,LIM S I.Microbial composition of the Korean traditional food“kochujang” analyzed by a massive sequencing technique[J].Journal of Food Science,2012,77(4):250-256.DOI:10.1111/j.1750-3841.2012.02656.x.
[15]PARK E J,CHUN J,CHA C J,et al.Bacterial community analysis during fermentation of ten representative kinds of kimchi with barcoded pyrosequencing[J].Food Microbiology,2012,30(1):197-204.DOI:10.1016/j.fm.2011.10.011.
[16]ROH S W,KIM K H,NAM Y D,et al.Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing[J].The ISME Journal,2010,4(1):1-16.DOI:10.1038/ismej.2009.83.
[17]PO?KA J,REBECCHI A,PISACANE V,et al.Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons[J].Food Microbiology,2015,46(4):342-356.DOI:10.1016/j.fm.2014.08.023.
[18]BOKULICH N A,THORNGATE J H,RICHARDSON P M,et al.Microbial biogeography of wine grapes is conditioned by cultivar,vintage,and climate[J].Proceedings of the National Academy of Sciences,2014,111(1):139-148.DOI:10.1073/pnas.1317377110.
[19]MARSH A J,O’SULLIVAN O,HILL C,et al.Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha(tea fungus)samples[J].Food Microbiology,2014,38(4):171-178.DOI:10.1016/j.fm.2013.09.003.
[20]ALEGRíAá,SZCZESNY P,MAYO B,et al.Biodiversity in Oscypek,a traditional Polish cheese,determined by culturedependent and-independent approaches[J].Applied and Environmental Microbiology,2012,78(6):1890-1898.DOI:10.1128/AEM.06081-11.
[21]SAKAMOTO N,TANAKA S,SONOMOTO K,et al.16S rRNApyrosequencing-based investigation of the bacterial community in nukadoko,a pickling bed of fermented rice bran[J].International Journal of Food Microbiology,2011,144(3):352-359.DOI:10.1016/j.ijfoodmicro.2010.10.017.
[22]王玉荣,孙永坤,代凯文,等.基于单分子实时测序技术的3个当阳广椒样品细菌多样性研究[J].食品工业科技,2018,39(2):108-112.DOI:10.13386/j.issn1002-0306.2018.02.021.
[23]RAVEL J,GAJER P,ABDO Z,et al.Vaginal microbiome of reproductive-age women[J].Proceedings of the National Academy of Sciences,2011,108(S1):4680-4687.DOI:10.1073/pnas.1002611107.
[24]XU N,TAN G,WANG H,et al.Effect of biochar additions to soil on nitrogen leaching,microbial biomass and bacterial community structure[J].European Journal of Soil Biology,2016,74(3):1-8.DOI:10.1016/j.ejsobi.2016.02.004.
[25]YUSOFF M Z M,HU A,FENG C,et al.Influence of pretreated activated sludge for electricity generation in microbial fuel cell application[J].Bioresource Technology,2013,145(11):90-96.DOI:10.1016/j.biortech.2013.03.003.
[26]CAPORASO J G,KUCZYNSKI J,STOMBAUGH J,et al.QIIMEallows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.DOI:10.1038/nmeth.f.303.
[27]CAPORASO J G,BITTINGER K,BUSHMAN F D,et al.PyNAST:a flexible tool for aligning sequences to a template alignment[J].Bioinformatics,2010,26(2):266-267.DOI:10.1093/bioinformatics/btp636.
[28]EDGAR R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.DOI:10.1093/bioinformatics/btq461.
[29]HAAS B J,GEVERS D,EARL A M,et al.Chimeric 16S rRNAsequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons[J].Genome Research,2011,21(3):494-504.DOI:10.1101/gr.112730.110.
[30]COLE J R,CHAI B,FARRIS R J,et al.The ribosomal database project(RDP-II):introducing myRDP space and quality controlled public data[J].Nucleic Acids Research,2007,35(1):169-172.DOI:10.1093/nar/gkl889.
[31]DESANTIS T Z,HUGENHOLTZ P,LARSEN N,et al.Greengenes,a chimera-checked 16S rRNA gene database and workbench compatible with ARB[J].Applied and Environmental Microbiology,2006,72(7):5069-5072.DOI:10.1128/AEM.03006-05.
[32]PRICE M N,DEHAL P S,ARKIN A P.Fasttree:computing large minimum evolution trees with profiles instead of a distance matrix[J].Molecular Biology and Evolution,2009,26(7):1641-1650.DOI:10.1093/molbev/msp077.
[33]R?YTI?H,MOKKALA K,VAHLBERG T,et al.Dietary intake of fat and fibre according to reference values relates to higher gut microbiota richness in overweight pregnant women[J].British Journal of Nutrition,2017,118(5):343-352.DOI:10.1017/S0007114517002100.
[34]LOZUPONE C,KNIGHT R.UniFrac:a new phylogenetic method for comparing microbial communities[J].Applied and Environmental Microbiology,2005,71(12):8228-8235.DOI:10.1128/AEM.71.12.8228-8235.2005.
[35]ZHANG J,GUO Z,XUE Z,et al.A phylo-functional core of gut microbiota in healthy young Chinese cohorts across lifestyles,geography and ethnicities[J].The ISME Journal,2015,9(2):1979-1990.DOI:10.1038/ismej.2015.11.
[36]ZHANG J,WANG L,GUO Z,et al.454 Pyrosequencing reveals changes in the faecal microbiota of adults consuming Lactobacillus casei Zhang[J].FEMS Microbiology Ecology,2014,88(3):612-622.DOI:10.1111/1574-6941.12328.
[37]ZHANG J,GUO Z,LIM A A Q,et al.Mongolians core gut microbiota and its correlation with seasonal dietary changes[J].Scientific Reports,2014,4:5001.DOI:10.1038/srep05001.
[38]GUO Z,ZHANG J,WANG Z,et al.Intestinal microbiota distinguish gout patients from healthy humans[J].Scientific Reports,2016,6:20602.DOI:10.1038/srep20602.
[39]周书楠,席修璞,董藴,等.琚湾酸浆面浆水细菌多样性评价[J].中国酿造,2018,37(1):49-53.DOI:10.11882/j.issn.0254-5071.2018.01.011.
[40]郭壮,沈馨,董蕴,等.襄阳大头菜腌制液膜醭细菌群落结构研究[J].中国酿造,2017,36(7):143-147.DOI:10.11882/j.issn.0254-5071.2017.07.031.
[41]SOGIN M L,MORRISON H G,HUBER J A,et al.Microbial diversity in the deep sea and the underexplored“rare biosphere”[J].Proceedings of the National Academy of Sciences,2006,103(32):12115-12120.DOI:10.1073/pnas.0605127103.
[42]YOUSSEF N,SHEIK C S,KRUMHOLZ L R,et al.Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNAgene-based environmental surveys[J].Applied and Environmental Microbiology,2009,75(16):5227-5236.DOI:10.1128/AEM.00592-09.
[43]MOSHER J J,BOWMAN B,BERNBERG E L,et al.Improved performance of the PacBio SMRT technology for 16S rDNAsequencing[J].Journal of Microbiological Methods,2014,104(11):59-60.DOI:10.1016/j.mimet.2014.06.012.