微生物多樣性基礎(chǔ)分析

OTU聚類與分類學(xué)分α多α多樣性分34OTU聚

OTU（OperationalTaxonomicUnits）/分類操作單（品系，種，屬，分組等）聚類標(biāo)

目 曲線及Shannon曲線）

OTUOTU聚類-步IdentifychimericChimeraOTUPickrepgenerateasetofrepresentativeOTUrepsCreatinganOTU軟件平臺(tái)-

tativeInsightsIntoMicrobialComparison ysisofmicrobialcommunitieshigh-throughputsequencingdatafromrawsequencingoutputthrough ysessuchOTUpicking,taxonomicconstructionofphylogeneticdownstream ysisproductionofpublication-quality OTUOTU聚類-QIIME步識別嵌合程序算法Blast_fragmentsapproachChimeraSlayer;去除嵌合程序描述removesequencesfromafastaorfastqfilebasedoninputOTU聚程序方法cd-hitLi&Godzik,2006;Li,Jaroszewski,&Godzik,Blast----Altschul,Gish,Miller,Myers,&Lipman,1990.Mothur----Schlossetal.,2009.prefix/suffix----Qiimeteam,unpublished.Trie----Qiimeteam,unpublished.uclust----Edgar,RCuclust_ref----Edgar,RCusearch61----Edgar,RC2010,versionv6.1.544usearch61_ref----Edgar,RC2010,versionv6.1.544OTU聚類-工具-High-throughputsearchandUSEARCHisaunique ysistoolwiththousandsofusersworld-USEARCHofferssearchandclusteringalgorithmsthatareoftenordersoffasterthanImprovedproductivityandUSEARCHcombinesmanydifferentalgorithmsintoasinglepackagewithationandThiscutsyourlearningcurve,reducesthenumberofstepsyouneedtotakeforgiventask,andslashescomputeUSEARCHwillencourageyoutoexploreyourdata,enablingnewinsightsandsuggesting ysesthatyoumightnothavetriedwithslowertools.FreeformostLicensestouse32-bitUSEARCHareofferedatnochargeforallusers, 分類學(xué)

ReleaseSILVASILVAprovidesalignedsmall(16S/18S,SSU)andlargesubunit(23S/28S,LSU)ribosomalRNA(rRNA)sequencesforallthree soflife(Bacteria,ArchaeaandEukarya).RibosomalDatabaseProject(RDP)RibosomalDatabaseProject(RDP)providesribosomerelateddataandservicestothescientificcommunity,includingonlinedata ysisandalignedandannotatedBacterialandArchaealsmall-subunit16SrRNAsequences.Thegreengeneswebapplicationprovidesaccesstothecurrentandcomprehensive16SrRNAgenesequencealignment.UNITEprovidesunifiedwayhowyoudelimit,identify,communicateandworkwithDNAbasedHypotheses(SH).AllSHsareconnectedtothetaxonnameand Sequencedataon- Release 16S,18S,23S, Release Release Functional Functional Release分分類學(xué)分類學(xué)數(shù)據(jù)

s s

dBacteria;pProteobacteria;cGammaproteobacteria;oB38;fnorank;gnorank;sOryza_sativa_Indica_GroupdBacteria;pProteobacteria;cAlphaproteobacteria;oRhizobiales;fPhyllobacteriaceae;gCohaesibacter;dBacteria;pProteobacteria;cAlphaproteobacteria;oRhodobacterales;fRhodobacteraceae;gdBacteria;pActinobacteria;cCoriobacteriia;oCoriobacteriales;fCoriobacteriaceae;gCollinsella;sdBacteria;pFirmicutes;cBacilli;oLactobacillales;fStreptococcaceae;gStreptococcus;sdBacteria;pProteobacteria;cAlphaproteobacteria;oRhodobacterales;fRhodobacteraceae;gSagittula;s dBacteria;pProteobacteria;cAlphaproteobacteria;oAdriatic90;fnorank;gnorank;suncultured_alpha_proteobacteriumss

dBacteria;pElusimicrobia;cElusimicrobia;oLineage_I_Endomicrobia;fnorank;gnorank;sdBacteria;pProteobacteria;cBetaproteobacteria;oBurkholderiales;fComamonadaceae;gdBacteria;pFirmicutes;cBacilli;oBacillales;fBacillaceae;gBacillus;sdBacteria;pActinobacteria;cActinobacteria;oStreptomycetales;fStreptomycetaceae;g dBacteria;pFirmicutes;cNegativicutes;oSelenomonadales;fVeillonellaceae;g dBacteria;pFirmicutes;cBacilli;oBacillales;fPaenibacillaceae;gPaenibacillus;s----根據(jù)silva庫中的參考序列對OTU進(jìn)行種屬鑒method=rdpThebayesianmethodlooksatthequerysequencekmerbycalculatesthecalculatestheprobabilityasequencefromagiventaxonomywouldcontainaspecifickmerQuery

calculatestheprobabilityaquerysequencecalculatestheprobabilityaquerysequencewouldbeinagiventaxonomybasedonthekmersitcontains實(shí)踐操實(shí)現(xiàn)-去嵌合QIIME法識別嵌合體序①使用參考序identify_chimeric_seqs.py-itrim.fasta-musearch61-ocheck_chimeras-#數(shù)據(jù)庫選擇細(xì)菌②無參考序列（功 和ITS可用此方法identify_chimeric_seqs.py-itrim.fasta-musearch61-ocheck_chimeras--wc-l #統(tǒng)計(jì)嵌合體序列實(shí)實(shí)現(xiàn)-OTU聚軟件QIIME法pick_otus.pyimeta.fasta-musearch61-s0.97-o OTU聚類cd #進(jìn)入OTU聚類文件lessmeta_otus.txt|awk'{if(NF>1){print}}'catmeta_otus_filter.txt|awk'{$1="OTU"NR;print$0}'|sed's//\t/g'pick_rep_set.py-iotu_seqids.txt-f../meta.fasta-mmost_abundant-o取OTU代表序make_otu_table.pyi -o #生成biom格式的表biomconvertiotu_table.biom-ootu_table.txt--table-type"otutablebcatotu_table.txt|sed-n'2p'|sed's/#//'catotu_table.txt|sed-n'3,$p'|sort-V|sed#生成OTU見的xls實(shí)實(shí)現(xiàn)-分類學(xué)注QIIME法 -mrdp-i -c0.8-/home/majorbio1/diversty/database/16s.silva.fasta-/home/majorbio1/diversty/database/16s.silva.tax-o&

--rdp_max_memory#用OTU代表序列進(jìn)行分類#生成biom格式的otu及分類biomconvert-iotu_taxa_table.biom-ootu_taxa_table.txt--header-keytaxonomy table-type"otutable"-b catotu_taxa_table.txt|sed-n'2p'|sed's/#//'catotu_taxa_table.txt|sed-n'3,$p'|sort-V|sed's/\.0//g'#生成OTU見的xlssummarize_taxa.py-iotu_taxa_table.biom-otax_summary_a-L1,2,3,4,5,6,7-asummarize_taxa.py-iotu_taxa_table.biom-otax_summary_r-L1,2,3,4,5,6,7#生成各分類水平統(tǒng)計(jì)結(jié)果文otu_table.biom:biom格式otu物種分類表。biom（BiologicalObservationMatrix）格式是生物學(xué) 結(jié)果結(jié)果文1 12α多樣32α多樣34生生物多樣Whichsamplehasthehighestdiversityα多index，Simpson'sdiversityindex.βShannonspeciesspeciesspeciesspecies式中S為物種數(shù)目，Pi為屬于種i 在全 中的比例際測量出的OTU數(shù)目i個(gè)OTU含有的序列數(shù)N有的序列數(shù) Simpson's 就為P^2。如果 α多樣性評mothur:summary.single-calculateavarietyofα-diversitycalculatorseachOTUChao-theChao1estimatorAce-theACEestimatorChao-theChao1estimatorAce-theACEestimatorShannon-theShannon-theShannonSimpson-theSimpson CoverageCoverage-theGood’s稀釋性曲稀釋性曲mothur:rarefaction.single-generateararefactioncurveusingavarietyofdiversity Sn=AveragenumberofOTUsobservedafterdrawingnindividuals.St=TotalnumberofOTUsinsampleofNtotalindividualsShannon曲

Shannon-Wiener曲mothur:

實(shí)踐操實(shí)實(shí)軟件otu2shared.pliotu_table.xls-l0.97-ometa.shared#多樣性rarefaction.single(shared=meta.shared,calc=sobs-chao-shannon,groupmode=f,#稀釋性曲線及Shannon-Wiener曲線等實(shí)mkdirmvmeta.*.summarymeta.*.rarefactionmeta.*.rabundmeta.*.r_chaometa.*.r_shannon#生成文件夾并將多樣性指數(shù)及稀釋性曲線及Shannon-Wiener曲線等相關(guān)文件移入文cdcp/home/majorbio1/diversty/scriptsshannon.R#進(jìn)入rabund文件夾并將rarafaction及shannon畫圖程序copy過Rscriptshannon.R#運(yùn)行程序畫rarefaction曲線及shannon曲shannon-ace-table.pl-d.-o#將*.summary文件整合成.html格式的表格,生成多樣性指數(shù)統(tǒng)結(jié)果文結(jié)果文結(jié)果文α多樣性-R作軟件：R ：#mycol<-c(34,51,142,23,50,27,31,75, plot(x=data$numsampled,y=data$X0.97,xlim=c(0,110),ylim=c(0,56.1),xlab="NumberofReadsSampled",ylab="RarefactionMeasure:rarefaction",type="l",col="white",font.lab=3,font.lab=2,sub="label:0.97",cex=0.7)leng<- points(x=data$numsampled,y=data$X0.97,type="l",col=colors()[51],lwd=3)leng<-length(data$numsampled) α多α多樣性分34Rank-abundanceRank-abundance曲 物物種累積曲實(shí)踐操環(huán)境物種總體分作圖RorcdRscriptrankabundance.R#進(jìn)入上一級文件夾，將rankabundance.R程序copy進(jìn)當(dāng) ，并用otu_table.xls圖Rscript#將specaccum.R程序copy進(jìn)當(dāng) ，并用otu_table.xls畫圖實(shí)現(xiàn)軟件：