16S信息分析報(bào)告2-北京奧維森

北京奧維森基因科技16srDNA信息分析標(biāo)準(zhǔn)信息分析〔初級(jí)〕根本數(shù)據(jù)處理〔使用內(nèi)部撰寫的程序?qū)υ嫉臏y(cè)序數(shù)據(jù)進(jìn)展根本處理〕通過(guò)Illumina〔Mise進(jìn)展Paired-endreads(Q20,90%標(biāo)準(zhǔn)過(guò)濾)trimreads2100bp低質(zhì)量序列；每個(gè)樣品數(shù)據(jù)產(chǎn)出具體統(tǒng)計(jì)結(jié)果見(jiàn)下表：reads數(shù)據(jù)統(tǒng)計(jì)：#Samples#HQreads(total)#HQreads(mean±SD)CA17110,6516,509±2,175HC19163,6908,615±3,081LK13127,4169,801±2,858Total49401,7578,199±2,992Reads數(shù)太少〔23條〕49個(gè)去除barcode序列，引物序列及tags過(guò)濾通過(guò)COPE軟件ConnectingOverlappedPair-EnV1.2.3.端測(cè)序得到的成對(duì)reads組裝成一條序列。利用內(nèi)部編寫程序去除兩端barcode序列，引物序列。PairedEndReadsreadsoverlap〔19個(gè)堿基〕Tags；然后去掉barcode序列，引物序列。為了得到高質(zhì)量的TagsTags依據(jù)長(zhǎng)度過(guò)濾，去嵌合(這里等的意思就是依據(jù)拼接條件過(guò)濾:1,堿基的ASCIIvalue33的過(guò)濾掉。2.overlap191998%的過(guò)濾掉。3.去掉引物序列的時(shí)候，允許一個(gè)錯(cuò)配，錯(cuò)配多于一個(gè)的過(guò)濾掉。)1-2tags的具體信息SampleIDRawTagNumFinalTagnumHC11756017,319HC296729,604HC31805317,826HC41218112,107HC51155811,477HC81148811,404北京奧維森基因科技HC91635416,095HC102158421,270HC1179897926HC121156111,449HC132490924,660HC142297922,736HC152074720,549HC161485714,728HC172117121,002HC181070010,605HC191135911,247CA81620316,040CA101092510,560CA1182547,690CA1294799,053CA1479477,584CA1682218,093CA171066610,479CA181078710,651CA51634416,154CA960475,861CA131029010,165高級(jí)信息分析OUT及其豐度分析OUT統(tǒng)計(jì)Tags0.97qiime〔v1.8.0〕軟件將其聚類為用于物種分類的OTU(OperationalTaxonomicUnits)，統(tǒng)計(jì)各個(gè)樣品每個(gè)OTU中的豐度信息，OTU的豐度初步說(shuō)明白樣品的物種豐富程度。493029OTU，其SingletonsOTU〔1的OTU〕0，NonsingletonsOTU3029。4.樣品OUT統(tǒng)計(jì)SampleNameOTUsTagsHC154117,319HC22699,604HC353017,826HC421512,107HC5HC421512,107HC520611,477HC821411,404HC945516,095HC1060021,270HC1226211,449HC1329424,660CA1045310,560CA117107,690CA126509,053CA145197,584CA162408,093CA1733010,479CA1828910,651CA533616,154CA93475,861HC111427,926CA1326910,165表5 OTU統(tǒng)計(jì)IndexOTUnumNo.ofIndexOTUnumNo.ofOTUs3029Assignedtofamilies1,708Assignedtogenera1,172Assignedtospecies314No.ofOTUspersample368±147Minno.ofOTUspersample127Maxno.ofOTUspersample7190.97OTUR〔v3.1.1〕Venn圖可以展現(xiàn)多樣品共有和各自特有OTUOTUOTU所代表的物種，可以找出不同環(huán)境中的核心微生物。北京奧維森基因科技圖2-1OTUvenn分析。不同顏色圖形代表不同樣品或者不同組別，不同顏色圖形之間交疊局部數(shù)字為兩OTU個(gè)數(shù)。Venn2-5個(gè)樣品或組別。OUTPCA圖如下：R〔v3.1.1〕畫圖軟件PCA分析(PrincipalComponentAnalysis)，即主成分分析，是一種分析和簡(jiǎn)化數(shù)據(jù)集的技要方面。通過(guò)分析不同樣品OTU〔97%相像性〕組成可以反映樣品的差異和距離，PCA運(yùn)樣品可能表現(xiàn)出分散和聚攏的分布狀況。北京奧維森基因科技圖2-2基于OTU豐度的PCA分析。橫坐標(biāo)表示第一主成分，括號(hào)中的百分比則表示第一主成分對(duì)樣品差異的奉獻(xiàn)值；縱坐標(biāo)表示其次主成分，括號(hào)中的百分比表示其次主成分對(duì)樣品差異的奉獻(xiàn)值。圖中點(diǎn)分別表示各個(gè)樣品。不同顏色代表樣品屬于不同的分組。北京奧維森基因科技22 Coremicrobiome分析圖表都是通過(guò)qiime〔v1.8.0〕軟件得到的OTU數(shù)與樣本數(shù)的關(guān)系：圖2-3掩蓋全部樣本的微生物組。橫坐標(biāo)表示樣品占的比率，縱坐標(biāo)表示包含OUT的數(shù)目。coremicrobiome〔即掩蓋全部樣本的微生物組17OTUs，其物種2-1。OTUsOTUTaxonomylevelTaxonomyname400850GenusStreptococcus437590GenusCapnocytophaga368428Speciesdispar645710GenusCampylobacter417699GenusFusobacterium395972GenusStreptococcus381841GenusStreptococcus140702GenusPeptostreptococcus413823GenusGranulicatella645697GenusCampylobacter414306GenusNeisseria260777GenusFusobacterium2023GenusNeisseria21908GenusNeisseria645708GenusCampylobacter414422FamilyGemellaceae北京奧維森基因科技1212 Genus Granulicatella生物多樣性分析單個(gè)樣品簡(jiǎn)單性分析通過(guò)計(jì)算Shannonindex,Chao1index,Phylogeneticdiversity(PD,wholetree)和observednumberofspecies共四個(gè)指數(shù)來(lái)進(jìn)展生物多樣性分析。通過(guò)qiime〔v1.8.0〕軟件計(jì)算樣品的Alpha多樣性值并用R〔v3.1.1〕軟件做出相應(yīng)的稀釋曲線，盒型圖。16SrDNA序列中的各種OTU的相比照例，來(lái)計(jì)算抽取n個(gè)〔n小于測(cè)得Reads序列總數(shù)〕Tags時(shí)各Alpha指數(shù)的期望值，然后依據(jù)一組n值〔一般為一組小于總序列數(shù)的等差數(shù)列〕與其相對(duì)應(yīng)的Alpha指數(shù)的期望值繪制曲線。如樣品有供給分組信息，且每組樣品個(gè)數(shù)不小于3，將對(duì)組間的Alpha多樣性指數(shù)進(jìn)展差異分析。差異分析的檢驗(yàn)方法為秩和檢驗(yàn)，假設(shè)組數(shù)為2，承受兩樣品比較的WilcoxonRank-SumTet R中的wilcox.tes假設(shè)組數(shù)大于Kruskal-WallisTest〔R中的kruskal.test〕。最終利用Alpha多樣性指數(shù)繪制盒形圖。差異分析與作圖均通過(guò)R軟件〔v3.1.1〕進(jìn)展?；贠TUAlpha多樣性〔表2-Alphachao1多樣性估算指數(shù)是依據(jù)所測(cè)得的tagsOTU的數(shù)量以及相比照Pvalue(KWp-vaule(CA-p-vaule(CA-Lp-vaule(HC-#Alphamean(CA)mean(HC)mean(LK))Pvalue(KWp-vaule(CA-p-vaule(CA-Lp-vaule(HC-#Alphamean(CA)mean(HC)mean(LK))HC)K)LK)chao1488.2993557357.6225984422.5433110.10303420.048708660.3593680.2476438observed_species243.0764706161.2631579199.54615380.021409050.015426790.21165110.09132691PD_whole_tree16.4888923513.1660461115.232958770.037857420.016745530.38587990.06422109shannon3.7781270132.997886233.4041635860.003926270.011310790.21670590.02426458測(cè)品中微生物的北京奧維森基因科技n分析〔樣本不分組：圖2-4單個(gè)樣品內(nèi)的Alpha 多樣性n分析〔樣本分組：北京奧維森基因科技圖2-5每組樣品內(nèi)的Alpha 多樣性。圖中紅色,黃色,藍(lán)色線分別表示CA,HC,LK組的rarefaction分析結(jié)果圖2-6為組Alpha多樣性盒形圖Alpha5〔最5條線，特別值以“”標(biāo)出。北京奧維森基因科技AlphaShannonindexCA>LK>HCCA/HC有明顯差異(P=0.008,Student’sttest)CA/LK,HC/LK差異不顯著樣品間簡(jiǎn)單度比較分析Beta多樣性〔Betadiversity〕分析是用來(lái)比較一對(duì)樣品在物種多樣性方面存在的差異大小。本分析中通過(guò)QIIME〔v1.8.0〕軟件，承受迭代算法，分別在加權(quán)物種分類豐度信息和75%Reads100次之后綜合統(tǒng)計(jì)得到最終的統(tǒng)計(jì)分析結(jié)果表及PCoA展現(xiàn)圖。Beta多樣性熱圖使用R〔v3.1.1〕軟件中的NMFaheatmap進(jìn)展作圖。UniFrac是通過(guò)利用系統(tǒng)進(jìn)化的信息來(lái)比較樣品間的物種群落差異一種衡量betadiversity越大。報(bào)告中給出的UniFrac結(jié)果分為加權(quán)UniFrac〔weightedUniFrac〕與非加權(quán)UniFirac〔unweightedUniFrac〕2種，其中weightedUniFrac考慮了序列的豐度，unweightedUniFrac不考慮序列豐度。從下面盒形圖看，CA組內(nèi)的物種豐度最大。WeightedUnifrac UnweightedUnifrac圖2-7 Beta多樣性的盒形圖北京奧維森基因科技Unifrac距離的主坐標(biāo)分析(PCoA)如下：WeightedUnifrac UnweightedUnifrac圖2-8Beta多樣性的主坐標(biāo)分析(PCoA)圖。假設(shè)兩個(gè)樣品距離越近，則表示這兩個(gè)樣品的組成越相像是否具有相像性。圖2-9UniFrac距離分布heatmapUniFrac結(jié)果的聚類，具有相像beta多樣性的樣品聚類在一起，反響了樣品間的相像性。物種組成分析本分析中分組后各水平的分類比較柱形圖是用QIIME〔v1.8.0〕軟件得到的，單個(gè)樣品的群落分布柱形圖和盒型圖是依據(jù)QIIME〔v1.8.0〕軟件計(jì)算的結(jié)果用R〔v3.1.1〕軟件畫的。北京奧維森基因科技樣品的群落分布圖，直觀的反響各樣品的群落組成。從門水平的群落分布圖中可以看出，F(xiàn)irmicutes，Proteobacteria。門(phylum)水平比較2-10分組后門水平的分類比較CA,HC,LK的物種組成。2-11樣品的門水平群落分布圖綱(class)北京奧維森基因科技2-12分組后綱水平的分類比較。CA,HC,LK的物種組成。2-13樣品的綱水平群落分布圖屬(genus)水平比較北京奧維森基因科技2-14樣品的屬水平群落分布圖25個(gè)屬的物種組成如下：可以看出，這些樣本中含量最高的屬為Streptococcus,Neisseria,Neisseriaceae(family),Campylobacter,Bacillus,Gemellaceae,TM7-3北京奧維森基因科技多組樣本的比較分析下面的表格都是通過(guò)QIIME〔v1.8.0〕軟件計(jì)算出的，熱圖是用R〔v3.1.1〕軟件畫的。2.3.4.1.1OTU水平的比較分析下表是在不同組樣本間有顯著差異的〔,s，共5個(gè)OTUPvalueCA_meanHC_meanLK_meanLineage10825390.00424330.30960880.46149550.3054158s_Streptococcus_infantis10340520.01148961.334E-059.02E-053.309E-05s_Streptococcus_infantisCU.OTU36090.0468380.00077174.302E-057.37E-05s_Streptococcus_infantisCU.OTU39510.04872320.00093470.00017980.0004562s_Streptococcus_infantis5615370.00460564.506E-052.515E-050.0001385s_Selenomonas_noxia27142670.00220230.00024412.728E-053.53E-05s_Prevotella_tannerae9686750.0009910.00145830.00210670.0067938s_Haemophiluspara_influenzae1688170.00580284.681E-057.871E-050.0002216s_Capnocytophaga_ochraceaCU.OTU15120.01886760.0001715.314E-054.729E-05s_Campylobacter_rectusCU.OTU42480.02630483.144E-053.67E-050.0001205s_Actinobacillus_porcinusCU.OTU46690.01292220.000336700.0006268o_LactobacillalesCU.OTU28840.02085850.00030420.00025670.000887o_Gemellales9319500.02998470.0003360.0005880.0003561g_Streptococcus43203170.04066871.213E-057.16E-050.0001015g_Streptococcus44167630.02667630.00040812.432E-054.272E-05g_Streptococcus2699070.01923650.00296490.00026950.000376g_Prevotella3245320.04713650.00020892.607E-055.232E-05g_Leptotrichia43057910.003092108.912E-050.0002187g_Cardiobacterium42949540.0205540.00057660.00043790.001282g_Capnocytophaga10103290.03950950.00023727.529E-050.0001302g_Capnocytophaga10986550.02105580.012873900.1271655g_BacillusOTU190.0272430.00114440.00373780.0004883g_Abiotrophia43211360.0378650.00039060.00344990.0012684f_StreptococcaceaeCU.OTU44370.01766786.529E-055.103E-060.000202f_StreptococcaceaeOTU20.00418114.005E-059.346E-060.0001607f_PasteurellaceaeCU.OTU38810.03384980.00021416.821E-050.0004476f_NeisseriaceaeCU.OTU20270.04195970.00012438.193E-050.0002474f_Neisseriaceae11016690.03959570.01894740.04067760.01988f_GemellaceaeCU.OTU1600.02701410.00017688.61E-063.201E-05f_Clostridiaceae8517040.03049670.00819870.00065750.0014748f_Clostridiaceae10900596.38E-056.461E-058.711E-050.0003815f_Carnobacteriaceae9497890.00371860.00126510.00208510.003773f_Carnobacteriaceae10659740.01214450.0024050.00456510.0070174f_CarnobacteriaceaeOTU100.02341810.000186800.001004c_BacilliCU.OTU310.0286421 0.0004367 2.232E-05 3.781E-05CU.OTU310.0286421 0.0004367 2.232E-05 3.781E-05p_Firmicutes2.3.4.2屬水平的比較分析個(gè)屬或科：CA_meanHC_meanLK_meanPvalue個(gè)屬或科：CA_meanHC_meanLK_meanPvalueg_Streptococcus0.43300460.60816750.39466440.001482408g_Campylobacter0.064858410.029354620.034725140.03853575g_Bacillus0.0129713900.12795236.82332E-05f_Gemellaceae0.023597830.045285970.022507560.0136056f_Carnobacteriaceae0.003761120.0067681990.011273880.0106726g_Haemophilus0.0030465010.0023524870.0077057340.0143493g_Lautropia0.0010965750.0047181260.0009672860.01870962g_Abiotrophia0.0011981370.0037715340.0004882660.02844294g_Acti