1、承擔(dān)單位：福建省農(nóng)科院農(nóng)業(yè)牛物資源研究所試驗(yàn)設(shè)計(jì)：劉國(guó)紅試驗(yàn)項(xiàng)目：采用rdp數(shù)據(jù)庫(kù)進(jìn)行芽抱桿菌菌種鑒定試驗(yàn)人員：王子旋、劉國(guó)紅試驗(yàn)負(fù)責(zé)：林營(yíng)志報(bào)告日期：2010-08-30計(jì)數(shù)月份：2010年08月研究資格系數(shù)（0.5-1.0）實(shí)驗(yàn)設(shè)計(jì)系數(shù)（0.9-1.0）報(bào)告得分|總分實(shí)驗(yàn)設(shè)計(jì)實(shí)驗(yàn)記載實(shí)驗(yàn)分析實(shí)驗(yàn)簡(jiǎn)報(bào)實(shí)驗(yàn)文章1-20%21-40%41-60%61-80%81-100%1%-5%設(shè)計(jì)不足，實(shí)施錯(cuò)誤6%-10%設(shè)計(jì)不全，實(shí)驗(yàn)無(wú)果11%-15%設(shè)計(jì)可以，實(shí)施不全16%-20%設(shè)計(jì)完整，分布實(shí)施21%-25%實(shí)驗(yàn)不全，記栽無(wú)果26%-30%實(shí)驗(yàn)不足，記栽不全31%35%獨(dú)立實(shí)驗(yàn)，記栽不全36%-40

2、%分布實(shí)驗(yàn)，記載完整41%-45%目的明確，實(shí)驗(yàn)完成46%-50%實(shí)驗(yàn)完整，記栽清晰51%-55%實(shí)驗(yàn)系統(tǒng)，結(jié)果可依56%-60%實(shí)驗(yàn)創(chuàng)新，分析溝晰61%-65%實(shí)驗(yàn)創(chuàng)新.結(jié)論淸晰66%-70%實(shí)驗(yàn)完整.統(tǒng)計(jì)淸晰71%-75%實(shí)驗(yàn)系統(tǒng)，數(shù)據(jù)可靠76%-80%報(bào)告完整，文章維形81%-85%結(jié)構(gòu)合理，分析冇據(jù)86%-90%邏輯清晰，統(tǒng)計(jì)合理91%-95%圖表完整，寫(xiě)作流暢96%-100%文獻(xiàn)完整，發(fā)衷水平福建省農(nóng)業(yè)科學(xué)院農(nóng)業(yè)生物資源研究所農(nóng)業(yè)微生物研究中心電話(huà)：0591 -87882571,傳真：0591 -878825711試驗(yàn)?zāi)康膔db核糖體數(shù)據(jù)庫(kù)項(xiàng)目位于http:/nlp.cme.msu.

3、edu/,位于密執(zhí)安州立大學(xué)的微生物生態(tài)學(xué)【i【心， 向科學(xué)界提供核糖體相關(guān)數(shù)據(jù)和服務(wù)，包括在線(xiàn)數(shù)據(jù)分析、構(gòu)建rna進(jìn)化樹(shù)和進(jìn)行rrna序列比 對(duì)等。該項(xiàng)目由美國(guó)國(guó)家科學(xué)基金會(huì)、美國(guó)能源部和密執(zhí)安州立大學(xué)發(fā)起支持于1989年開(kāi)始，到 2010年已升級(jí)到版本10。截止2010年08月14日，數(shù)據(jù)庫(kù)容量達(dá)到了 1,379,424條。rdp 數(shù)據(jù)庫(kù)項(xiàng)目包括 myrdp, hierarchy browser, sequence cart, tree builder, sequence match, probe match, classifier等。myrdp提供創(chuàng)建帳戶(hù)、上傳文件、篩選rdp序列、融合

4、比較、數(shù)據(jù)下載 和數(shù)據(jù)共亨服務(wù)；hierarchy browser提供分類(lèi)樹(shù)瀏覽、序列選擇、搜索和高級(jí)搜索、數(shù)據(jù)下載服務(wù)； sequence cart保存用戶(hù)的序列選定情況，相當(dāng)于購(gòu)物車(chē)；tree builder提供進(jìn)化樹(shù)構(gòu)建功能，可采用 私冇數(shù)據(jù)和公用數(shù)據(jù)；sequence match捉供序列比對(duì)功能；probe match提供探針比對(duì)功能；classifier 提供物種分類(lèi)功能，包括序列上傳、調(diào)整顯示深度和可信度等。本報(bào)告主要介紹使用rdp進(jìn)行芽抱桿菌種類(lèi)鑒定的使用方法步驟。2方法步驟以本實(shí)驗(yàn)室測(cè)定的一株芽抱桿菌16s序列為樣本進(jìn)行說(shuō)明rdp的使用步驟。2.1樣本我們以一段芽范桿菌的16s

5、序列為例說(shuō)明如何使用該系統(tǒng)。樣本序列如下：1tacgactcgtcctcggttcccggcgatcctctggagattacggctaccttgttacgactt61caccccaatcatctgtcccaccttcggcggctggctccaaaaggttacctcaccgacttc121gggtgttacaaactctcgtggtgtgacgggcggtgtgtacaagacccgggaacgtattca181ccgcggcatgctgatccgcgattactagcgattccggcttcatgtaggcgagttgcagcc241tacaatccgaactgagaatggttttat

6、gggattggcttaacctcgcggttttgcagccct301ttgtaccatccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacg361tcatccccaccttcctccggtttgtcaccggcagtcaccttagagtgcccaactgaatgc421tggcaactaagatcaagggttgcgctcgttgcgggacttaacccaacatctcacgacacg481agctgacgacaaccatgcaccacctgtcactctgtccccgaagggaaccttctatctcta541gaagtagcagag

7、gatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccac601atgctccaccgcttgtgcgggtccccgtcaattcctttgagtttcagtcttgcgaccgta661ctccccaggcggagtgcttaatgcgtttgctgcagcactaaagggcggaaaccctctaac721acttagcactcatcgttacggcgtggactacagggtatctaatcctgttcgctccccacg781ctttcgcgcctcagcgtcagttacagaccagagagccgccttcgccactggtgttcctcc

8、841acatctctacgcatttcaccgctacacgtgaattccgctctcctcttctgcactcaagtt901ccccagtttccaatgaccctccacggttgagccgtgggctttcacatcagacttaaggaa961ccgcctgcgcgcgctttacgcccaataattccggacaacgcttgccacctacgtattacc1021gcggctgctggcacgtagttagccgtggctttctggttaggtaccgtcaaggtacctgct1081tatttaacaggtacttgttcttccctaacaacagagctttacg

9、atccgaaaaccttcatc1141actcacgcggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcc1201tcccgtaggagtctgggccgtgtctcagtcccaatgtggccgatcaccctctcaggtcgg1261ctacgcatcgtcgccttggtgagccgttacctcaccaactagctaatgcgccgcgggccc1321atccttgagtggtagctaaaagccaccttctaacatatcctcatgcgaagatatgtgtta1381tccggtattagctccggtttccc

10、ggagttatccccgtctcaagggcaggttgcccacgtg1441ttactcacccgtccgccgctaagttttgaaaagcaagcttttcaaaactccgctcgactt1501gcatgtatta ggcacgccgc cagcgttcgt cctgagccag gatcaaactc taatcgtcca1561ctgccgtcat gctc序列采用fasta格式保存，示例文件名采用16s.txt,用記事本打開(kāi)如下所示。>20100828tacgactcgtcctcggttcccggcgatcctctggagattacggctaccttgttacgact

11、tcaccccaatcatctgtcccaccttcggcggctggctccaaaaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaagacccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccggcttcatgtaggcgagttgcagcctacaatccgaactgagaatggttttatgggattggcttaacctcgcggttttgcagccctttgtaccatccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgac

12、gtcatccccaccttcctccggtttgtcaccggcagtcaccttagagtgcccaactgaatgctggcaactaagatcaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtcactctgtccccgaagggaaccttctatctctagaagtagcagaggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggtccccgtcaattcctttgagtttcagtcttgcgaccgt

13、actccccaggcggagtgcttaatgcgtttgctgcagcactaaagggcggaaaccctctaacacttagcactcatcgttacggcgtggactacagggtatctaatcctgttcgctccccacgctttcgcgcctcagcgtcagttacagaccagagagccgccttcgccactggtgttcctccacatctctacgcatttcaccgctacacgtgaattccgctctcctcttctgcactcaagttccccagtttccaatgaccctccacggttgagccgtgggctttcacatcagacttaagga

14、accgcctgcgcgcgctttacgcccaataattccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggttaggtaccgtcaaggtacctgcttatttaacaggtacttgttcttccctaacaacagagctttacgatccgaaaaccttcatcactcacgcggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcctcccgtaggagtctgggccgtgtctcagtcccaatgtggccgatcaccctctcaggtcg

15、gctacgcatcgtcgccttggtgagccgttacctcaccaactagctaatgcgccgcgggcccatccttgagtggtagctaaaagccaccttctaacatatcctcatgcgaagatatgtgttatccggtattagctccggtttcccggagttatccccgtctcaagggcaggttgcccacgtgttactcacccgtccgccgctaagttttgaaaagcaagcttttcaaaactccgctcgacttgcatgtattaggcacgccgccagcgttcgtcctgag ccaggatcaaactctaatcgtc

16、cactgccgtcatgctc2.2 myrdp 功能打開(kāi)網(wǎng)站首頁(yè)，在myrdp功能處，新建一個(gè)用戶(hù)。登錄賬戶(hù)名采用email形式（應(yīng)全部小寫(xiě)形 式），密碼通過(guò)email發(fā)送回來(lái)。登陸后選擇upload功能，將剛才的文件上傳。上傳后myrdp如下圖所示。23 rdp classfier 模塊打開(kāi)首頁(yè)。explore our online analysis tools:選擇 classifier, rdp clissfer 基于 nomenclatural taxonomy and bergey's manualo 將待測(cè)序列粘貼 到輸入框中，或者做成fasia或genbank格式的文

17、本文件，上傳，單擊”submit”執(zhí)行分類(lèi)比較，即得 到所需的結(jié)果。如果建立了 myrdp,也可以選擇已上傳的數(shù)據(jù)。classifier startvideo tutoridl i help jintroductionuse our dassifier to assign 16s rrma gene sequences to the new phytogeneticauy consistent higher-order bacterial taxonomy hierarchical taxa is based on a naive bayesian rrna classifier. the x

18、wty trained dassifier allows dassification of both bacterial and ardiaeal 16s rrna sequences. cussiffcation algorithm has been published. please cite: wang, q, g m. garrity, j. m. tledje, and j. r. cote. 2007. halve bayesian classifier for rapid assignment of rrna sequences into the new bacterial ta

19、xonomy. appl environ mkrobiol. 73(16):5261-7. the classifier command line program. along with the source code .javadoc example taxonomy and sequence files, and help files is freely avahablc from sourceforge and h released under the terms of tbc gnu general putmk licensedownload the command-line prog

20、ram rdp aulticlassifier. this program uves rdp classifier on multiple samples. besides the output for detail classification assignments the program summarizes the assignment count for each taxon. additional options are described in the readme along with instructions for us“help topics: taxonomic hie

21、rarchy model and classification algorithmpartial sequences with length shorter than 2!>0 bps should use bootstrap cutoff 5哄please enter your sequences:1 sequences selected from myrdp account.dowilh so3c2d soquonc” |choose a file to upload: |迭擇文件耒送將文件cut and paste sequence(s) (in fasta, genbank, o

22、r em8l format):通過(guò)classfier可以得到一個(gè)大致的鑒定結(jié)果，但這還不夠。classifier : hierarchy view(start over | assignment detail | help classifier: rdp naive bayeshn rrha classifier version 2.2, march 2010taxovmxnical hierarchy: rdp training set 6. based on nomendatural taxonomy and bergey s manualquery file:query submit d

23、ate: fri aug 27 09:58:10 eot 2010display depth: 110confidence threshold: 195% | refresh |domain%librarybacteria100.0hierarchy viwr (dick a node to makf 9t tht root - click iht root(o sequence ossfyxnenc detail):norank root (1 sequences) show assament detail download hierarchy as text file domain bac

24、teria (1)» » * phylum "firmicutes* (1)» - » * dass bacihr (1)» » * order bacillales (1) famity baciuaceae (1)» « » * genus bdciuu、(1)quesbons/comments: rdpsu2.4 seq match比對(duì)功能在正式的比較中，我們可以用seq match功能檢索近緣種序列，然后用tree build功能構(gòu)建進(jìn)化 樹(shù)。seqmatch sta

25、rt(video tutorial | beip )did you know you can sc<|uenc<*$ from myrdp and hkrorchy browser to do seqmatd)? percent idendty scores will be reported for aligned sequeixes (limited to 2000).please enter your sequences:1 sequences selected from myrdp account.do seqmaich with selected sequenceschoo

26、se a file to upload: |玉擇文件|未詵樣丈件cyt and paste sequencw(s) (in fasta, genbank, or embl format): >20100828tacgicgtcctcggitccccccgatcctcrggjwattacggclaccttgtstrain: type© non type ® bothsource:uncultured'- isolates bothsize:# >1200©<1200© bothquality:° good一 suspec

27、t c bothtaxonomy: nomwkutufal © ncbikwn matches:20 esubmit | reset作為16s,我們選擇模式菌株，純培養(yǎng)菌株，長(zhǎng)度大于1200 bp。submit進(jìn)行比對(duì)。這和genbank 最大的區(qū)別在于可以要求在純培養(yǎng)的模式菌株屮選擇。數(shù)據(jù)可選擇已上傳的，也可以在此上傳新文 件，或者直接貼入序列。提交后需等待服務(wù)器的計(jì)算。等待中seqmatch : query sequences statusstatus: fundingcurrent tmie: fri aug 27 10:04:m eot 2010proqrcss; 8% co

28、mpletedrefresh cancel得到的比對(duì)結(jié)果。seqmatch : summary(new match | wniniary | hthp )select am match hits to seqcartdisplay depth: auto lineage (click node to return it to hierarchy view):hierarctiy view:no rank root (1) (query sequences) show prinur friondty wwfts download as text file |【eiom domain bacter

29、ia (1)phylum mflrmicutes* (1)dass (l)order bacilales (1)family 8adlaceae (1) genusbacwus (l) unknown view selectable matchesdau set options:strain: typenon type©bothsource:uncultured° isolatesbothsize:。>1200© <1200© bott)亠reesh單擊最末一級(jí)的結(jié)果，展開(kāi)并選擇所冇的相似菌株。seqmatch : detail hierar

30、chynew match | summary | detail | help j save selection and return to summaryquery sequence: u005450671120100828, 1480 unique oligosmatch hit format:short id, orientation, similarity score, s_ab score, unique common oligomers and sequence full name. more help is availablelineage:no rank root (20/20/

31、77j2) (selected/match/to tai rdp sequences) domain bacteria (20/20/7448) phylum -firmkutes- (20/20/1530)class "bdcillf (20/20/991) order baciuales (20/20/626) family bacillaceae (20/20/312)(20/20/162)59s000007628s000107717s000130255s00013129150003668525000373337s000390853s000478419s000429574s00

32、0434839s000437736s000447571s000528024s000598282s0006279135000842016s00084252479519lz7>l/l<l/l/l/ml<l/m7l/m!7vnolcakixated 0.785 1394 no匕calculated 0.800 1428 not.oilculated 0.796 1450 not_calcixated 0.797 1325 not.cakulated 0.816 1410 not.cakulated 0.801 1415 nolcalculated 0.739 1306 not.ca

33、kulated 0.979 1374 not.calculated 0.786 1437 nolcalculated 0.788 1418 calculated 0./91 1410 not.calculated 0.807 1417 no匕calcinated 0.795 1377 not.cakulated 0.793 1447 not.cakulated 0.807 1393 nolcateulated 0.800 1406 not.cakulated 0.785 1445 not calculated 0.788 1266not0.797 h01bacmius halmapalus (

34、t); dsm 8723; x76447bacillus carboniphilus (t)： xm9731; ab021182bacfllus addkoo (t); 105-2； af547709bacillus benzoevorans 仃)；ncimb 12555; x60611bacillus shacktetonii (t); lmg 18435; aj250318bacillus humi (t); type strain: img 22167; 527210bacilus koreensis 仃)；br030; ay667496bacillus endophyticus (i)

35、; 2dt; af295302bacillus slrath (t); 171544; af071856bacillus aquimaris (t); tf-12; af483625bacillus circulans (i); ay043084bacillus sporothermodurans (t); m215; u49078baciuus herbersteinensfa (t); type strain: d-l,5a; aj781029bacillus oleronius (t); atcc 700005; ay988598backlus niabensis (i); 4t19;

36、ay998119bacillus pocheonensis (t); gsor 420; ab245377bacillus isabeliae (t); type strain: cvs-8; am503357backlus canaveratius (t); ksc.sf8b; dq870688js firmus (tl: lam 12464; di 6268選擇所需的序列，然后點(diǎn)擊save selection and return to summary,我們的選擇就保存到seq cart 中了。通過(guò)此步驟，我們得到了和我們序列最相近的一批純培養(yǎng)模式菌株的序列，然后可以通過(guò)進(jìn) 化樹(shù)構(gòu)建的方法

37、獲得更準(zhǔn)確地判定。2.5 treebuild構(gòu)建進(jìn)化樹(shù)在上一步的步驟中，我們篩選到了和待測(cè)序列相似的近緣序列。下面進(jìn)一步記得選擇myrdp 屮的待測(cè)序列。ohtn fm mkkoeial kotoct8 michigan state universityoverviewgroup name (selected)submitter iddateprojecttotal pendingafu 20100818 (1)yingzhilin®i?27 au% 10bacillus1 0 1 00questions/ c omments: r dps uh 公 toptopac

38、tfckint or q selects (adds) - ok king q deselects (renvovesl for download and analysisada" - failed - undbgnedmkmcamtmt advancing knowledge « n i « i » 11 > lamfocminc lhc£ 1992-2009 michigan sute unspity board qf trustees east larxint ml 48824 aasu h an amrmatyve-actmn.

39、equal opportumty employer.單擊序列前方+號(hào)，挑選我們需要的序列，此時(shí)序列前面的標(biāo)志變成了號(hào)。察看序列選擇情況, 單擊seq cart,網(wǎng)頁(yè)列出了已選擇的序列信息。單機(jī)右側(cè)的download可將序列以fasta的格式下載到 本地。sequence cart(video tutorial | download ) your sequence cart oontakis 21 sequence.1 myrdp sequences, 1 aliened20 public sequences20 type strain0 environmental sequence(s)20

40、sequencefs) with length >= 1200 bases0 sequence(s) with low qualityreset cart|save your sequence cart (alisnad only) for future ust:rdpx&ciefia (21save cartretrieve an existing filt to current session:file contains:< rdp id o gefibank accno'choose upload:遇折文件:未選擇文件retrieve選擇tree build功

41、能，模型選擇rdpx-bacteria,數(shù)據(jù)組采用seq cart屮選擇好的序列，執(zhí)行create tree.等待中。tree builder start'ddeo tutorial help j(currently 0 task(s) are waiting in the queue.)your seq cart contains 21 aligned sequences:1 myrdp sequences,20 public sequencesselect sequences for treeing using the hierarchy browser, sequence mat

42、di and myfdpminimum com(>arable position is 200note: this task will take between 6 seconds and 45 minutes depending on how many sequences were selected and server load (during peroidsof extreme h加 server toad, you may not be able to submit requests).select alignment model:rdpx-bactena (21 seqs)0s

43、elect outgroup:(s000001659j bacillus halmapalus (t). 13crfatf trffaboutselect myftdp and/or public rdp sequences to create a phytogenetk tree using the weighbor weighted neighbor-joining tree building algorithm. the results are presented in an in teractive java applet that allows users to rearrange

44、nodes, and make other cosmetic dianges. the tree can be downloaded in nwidc format or as a postsalpt fitetree builder statusstatus:start time fri aug 27 10:30:42 eot 2010computing dkunce matrix.donerunning weighbor.current time: fri aug 27 10:30:43 eot 2010refresh cancelthis page will be automatical

45、/ refreshed every 5 seconds until tree is completed.questions/comments: rdpsu toptop構(gòu)建完成后在網(wǎng)頁(yè)中顯示結(jié)果，如對(duì)結(jié)杲不甚滿(mǎn)意，可在網(wǎng)頁(yè)中直接調(diào)整。也可將進(jìn)化樹(shù)以指 定格式下載。通常我們會(huì)用ctrl + m命令保存成mega格式的文件，在mega軟件中進(jìn)行修改。tree builder resultstart over | tree builder help tree commandsalt«cuck on a blue node to swap chmdren'<f

46、 key tousles distance dbpsy*b* key toggles bootstrap value (> 50% are high6$hted) sfmkebar toggles edit/prlnt nwlesaving treecontrol-s saves tree in newick formatcontrol*m saves tree in miga compatible newkk formatcontrol-p save, tree in ps format (use ps2p0f free online converter)*' and *

47、9; keys zoom in and out control-n shows display name control-d shows description control-! shows seqidnote: m applet does not reioond to keyboard commands or your web browser is receding the command% instead of the applet, cick fmide the tree view are« to retain focus.)l201008285771 bacillus er

48、dophyticus (t); 20t bacillus koreensis (t); br030 becillcs shackletonii (t); lmg 18435(bacillus oleronius (t); atcc 700005bacillus sporotnermodurans (t). m215bacii：us addicola (t)； 105-2 bacillus isabeliae (t); type strain: cvs-8 bacillus caroo rwpfiug (t); jcm9731 bacillus pocneonensis (t); gsoil 4

49、20bacillus nnnus (t); iam 124s4ibacillus nealsonn (t); dsm 15077薪bacil us ciirulans (t)bacillus 引 ralis (t). 171544 bacilus canaxeralius (t). ksc sf8b bacillus denzoevorans (t). ncimb q555 bacillus hertjersteinensis (t); type strain d-l.5a bac 卅 us n iabensls (t). 4t19 bacinus humi (t); type strain:

50、 lmg 22167 bacillus aqulmarls (t)： tf-12 bacillus hainapaius (t). dsm 8723treebuilder采用jukes-cantor模型計(jì)算距離矩陣，bootstrap值100,如果想換些參數(shù)構(gòu)建?？?以下載已選定的序列用mega或phylip等軟件進(jìn)行進(jìn)化樹(shù)構(gòu)建和分析。我們將序列下載后進(jìn)行了 兩端修剪，再用mega進(jìn)行進(jìn)化樹(shù)構(gòu)建。下圖是用mega 4.0繪制出的進(jìn)化樹(shù)，對(duì)數(shù)據(jù)進(jìn)行了修剪,比較策略是 nj > method(nj) > gap ( pairwise deletion) > method(nucl

51、eotide > jukes-cantor > test > bootstrap > replication(iooo),得到的進(jìn)化樹(shù)中待測(cè)序列與bacillus endophyticus最為接近，這個(gè)結(jié)果 和rdp網(wǎng)站的計(jì)算結(jié)果是相同的。99ay043084.1:1.1496 bacillus circulans.47 |eu656111.1:1.15o9 bacillus nealsonii.x60611.1:7. 1430 bacillus benzoewran. af071856.1:1.1523 bacillus siralis (.d16268.1:1.14

52、83 bacillus firmus (t)dq870688.1:1. 1323 bacillus canaveral.-ab245377.1:1.1495 bacillus pocheonen.ay667496.1:1.1370 bacillus koreensis.x76447.1:1. 1504 bacillus halmapalus .56aj781029.2:1. 1464 bacillus herberste.00828100ay998119.2:1.1478 bacillus niabensis.5302.1:1.-1470 bacillus endophyti.aj627210.1:1. 1504 bacillus humi (t).af483625.1:1. 1507 bacillus aquimaris.abo21182.1:1.15o4 bacillus carboniph.am503357.2:1.1523 bacillus isabeliae.5080 raf547209.1:1.