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SOME TRAINING ON NUCLEOTIDE SEQUENCES: EDITION, REGISTRATION, ALIGNMENT AND TREE BUILDING

SOME TRAINING ON NUCLEOTIDE SEQUENCES: EDITION, REGISTRATION, ALIGNMENT AND TREE BUILDING. Y.Ph. Kartavtsev A.V. Zhirmunsky Institute of Marine Biology of Far Eastern Branch of Russian Academy of Sciences, Vladivostok 690041, Russia, e-mail: yuri.kartavtsev48@hotmail.com. ГЛАВНЫЕ ВОПРОСЫ.

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SOME TRAINING ON NUCLEOTIDE SEQUENCES: EDITION, REGISTRATION, ALIGNMENT AND TREE BUILDING

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  1. SOME TRAINING ON NUCLEOTIDE SEQUENCES: EDITION, REGISTRATION, ALIGNMENT AND TREE BUILDING Y.Ph. Kartavtsev A.V. Zhirmunsky Institute of Marine Biology of Far Eastern Branch of Russian Academy of Sciences, Vladivostok 690041, Russia, e-mail: yuri.kartavtsev48@hotmail.com

  2. ГЛАВНЫЕ ВОПРОСЫ 1. Sequence edition and their registration in GenBank. 2. Data format and gene banks available. 3. Sequence alignment. 4. Finding an optimal model of nucleotide substitution. 5. Tree building with software packageMEGA-3 (MEGA-4). 6. Annotation onPAUP, MrBayesand some other programs.

  3. APPLICABILITY OF DIFFERENT DNA TYPESIN PHYLOGENETICS AND TAXONOMY Spacers [ITS-1, 2] mtDNA nDNA, rDNA SpeciesGenusFamilyOrderClassPhylum Most substantiated statistically results Statistically significant results

  4. МАТЕРИАЛ И МЕТОДЫ

  5. 1. SEQUENCE EDITION AND THEIR REGISTRATION IN THE GENBANK, NCBI (1) Original sequence that obtained from a sequencing machine requires an edition. Many requirement for the edition meet such program packages (PP) as MEGA-3 orMEGA-4 (http://www.megasoftware.net/), GeneDOC (http://www.nrbsc.org/) etc. Most suitable PP tool for the primary edition is Chromas (Chromas-pro, that is available at http://www.flu.org.cn/enorhttp://www.technelysium.com.au/chromas.html). Currently realized version (Chromas-pro 2.31) let to perform a number of edition options. • Opens chromatogram files from Applied Biosystems and Amersham MegaBace DNA sequencers. • Opens SCF format chromatogram files created by ALF, Li-Cor, Visible Genetics OpenGene, Beckman CEQ 2000XL and CEQ 8000, and other sequencers. • View Genescan genotype files. • Save in SCF or Applied Biosystems format. • Prints chromatogram with options to zoom or fit to one page. • Exports sequences in plaint text, formatted with base numbering, FASTA, EMBL, GenBank or GCG formats. • Copy the sequence to the clipboard in plain text or FASTA format for pasting into other applications. • Export sequences from batches of chromatogram files, with automatic removal of vector sequence. • Reverse & complement the sequence and chromatogram. • Search for sequences by exact matching or optimal alignment. • Display translations in 3 frames along with the sequence. • Copy an image of a chromatogram section for pasting into documents or presentations.

  6. 1. SEQUENCE EDITION AND THEIR REGISTRATION IN THE GENBANK , NCBI (2) Main task that CROMAS can perform is a comparisonof sequences, a removalof vector sequences in the beginning and in the end of chains, an inversion of the anti-parallel sequence (chains), a creation ofa consensus sequenceand recording all information in a mode that convenient for further calculations. Fig. 1.1 presents a view of sequences in CHROMAS PP editor. Fig. 1.1. A graphic and symbolic representationof a sequence fragment at cytochrome oxidase 1 (Со-1) gene in flounder, Liopsetta pinifasciata. Sequencing made with АBI-3100 (Applied Biosistems, USA) machine. Four repeated sequencesobtained with different primers (1K_F2 etc, left) and they are shown as peaks and their lettertranslation. After the inversion of the anti-parallel chains (1KR1_L_pand 1K_R2 etc) and performing their complementation sequences have automatically aligned. The consensus sequence that is under edition shown above. Chromatogram lines and letters of four nucleotides are shown in different colorfor better visual perception.

  7. 1. SEQUENCE EDITION AND THEIR REGISTRATION IN THE GENBANK , NCBI (3) • After an edition in CHROMAS or any other editora sequence of nucleotideshave to register it in a gene bank. For a registration of single genes or their segmentstheBankit utility is convenient.This utility let to submit a sequence or set of them in the interactive mode with the attribution to them a preliminary codes and after checking the codes of accession to the GenBank data base. In Fig. 1.2 there is a fraction of info that provided under request in the GenBank site. Fig. 1.2. Fragment of theGenBank window. Data are shown for the complete mtDNA genomeof one flatfish species (Pleuronectiformes).

  8. 2. DATA FORMAT AND GENE BANKS AVAILABLE • The submitted sequences will be accessible for overall usage after agreed date, usually after 1 year and publication of a paper. Particular sequence is accessible in different formatsGenBank, FASTAetc. In the first case it is looks likeas below (Fig. 2.1). 1 gtgcctgagc cggaatagtc ggggacaggc ctaagtctgc tcattcgagc agagctaagc61 caacctgggt gctctcctgg gagacgacca aatttataac gtaatcgtca ccgcacacgc121 ctttgtaata atcttcttta tagtaatacc aattatgatn cggagggttc ggaaactgac181 ttattccatt aataattggg gcccccgnat atggccttcc ctcgaataaa taacatgagt241 ttctgacttc tacccccatc ctttctcctc cttctagcct cttcaggncg tcgaagctgg301 ggcagggaca ggatgaaccg tgtatccccc actagctgga aatctagcac acgccggagc361 atcggtagac ctcaccattt tctctcttca ccttgccgga atttcatcaa ttctaggggc421 aatcaacttt attactacta tcatcaacat gaaaccaaca gcagtcacta tgtaccaaat481 cccactattt gtctgagccg tactaatcac cgcacgtcct tcttcttctt tcacactacc541 acgtcactgg ccgctggcat tacaatgcta ctgactagac cgcaacacta aacacaaaca601 cttctttgac cctgcyg Fig. 2.1. Partial nucleotide sequence Со-1 gene in flounder, Pseudopleuronectes obscurus. In the left column ordering numbers for first nucleotides are shown. Nucleotides are grouped by 10with total number 60 in a row. Other info in the NCBI window was shown above (Fig. 1.2). For a sequence registration one of three most recognized gene banks available: NCBI (USA), DDBJ (Japan), and EMBL (EU). These three banks are connected and exchange data. Thus, made a registration (submission) of a sequence, for instance inthe GenBank (http://www.ncbi.nlm.nih.gov), an author granted a confidence from an unwanted access in a certain agreed time and then these sequences become available to any user of Internet. • You are also free for a submission of your data in the European DNA bank, EMBL (http://www.ebi.ac.uk/embl/ ), or in the DNA data bank of Japan, DDBJ (http://www.ddbj.nig.ac.jp/searches-e.html ). There are also local DNA data banks, e.g. the Japan Center of BioResources, RIKEN (http://www.brc.riken.jp/lab/dna/en/), the North Bank, NGB (http://www.ngb.se) etc.

  9. 3. SEQUENCE ALIGNMENT (1) • Sequence alignment(выравнивание) is very important procedure, which anticipates theirquantitative analysis including a calculation of similarity-distances measures, homology estimate, and at lastbuilding different molecular phylogenetic trees (dendrograms). There are several algorithms of alignment that performed by different , sequence processors (editors). We will consider here for short only one sequencealignment that makeCLUSTAL W, a program adopted forOSWindows. • For the alignment you should first load the sequences into the editor. There are 3 way to do this: (1) Making a directrecord of nucleotide sequences one by one in a consequent window of the editor, (2) Importingthe sequences from a filethat was prepared before, and(3) Copying a sequence via clipboard from former editor to CLUSTAL W window. In Fig. 3.1 the interface of the CLUSTAL W editor is shown (Thompson et al. 1994), that integrated withMEGA;cases before (А) and after (В) alignment. А

  10. 3. SEQUENCE ALIGNMENT (2) В Fig. 3.1. Windows of the CLUSTAL Walignment editor (Alignment explorer) inMEGA, with fragments of Сyt-b gene nucleotide sequencesfrom several fish species before (А) and after alignment completed (В). With same color similar sitesare shown. An asteriskmarks sites that has 100% homology of nucleotides, i.e., these nucleotides are identical in all the sequences in a set. After the species names other identifiers (Labs’ codes or GenBank accession numbers) are denoted.

  11. 3. SEQUENCE ALIGNMENT (3) • In the above case the sequences were loaded via clipboard (Fig. 3.1). Make run of MEGA-3 (MEGA-4), we can chouse in the main menu: Alignment  Alignment explorer/Clustal  Create a new alignment («выравнивание»  «редактор выравнивания/Clustal» «создать новое выравнивание»). • In the last options there are actually 3 possibilities: Create a new alignment («создать новое выравнивание»), Open a saved alignment session («открыть сохраненную сессию выравнивания»), Retrieve sequence from a file («вывести последовательность из файла»). • When sequences are loaded, an author meets, as a rule, with a dimension problem: sequences length is unequaland their starts & endsare not complemented;more over, many sequences have deletions/insertions (Gaps), which are not coincide in different individuals and species. Alignment allows to solve all these problems.

  12. 3. SEQUENCE ALIGNMENT (4) Technically, to start CLUSTAL W execution you have to chooseall sequences and run the option“Alignment” of the main menu. As a result of this action a special dialog box appeared (Fig. 3.2). In Fig. 3.2 two dialog boxes are shown that suits for certain setting under alignment, which proceeds in the two steps. Fig. 3.2. Dialog boxes of the MEGA integrated CLUSTAL W editor that helps to perform alignment in an appropriate and user specified mode.Opened windows are for setting the penalty options (Penalties) underpair-wise alignment (Pairwise Parameters) and multiple alignment (Multiple Parameters).

  13. 3. SEQUENCE ALIGNMENT (5) • Pushing the button execute (ОК) execute alignment. The alignment is a delicate art and may take patience. Different sets of sequences takes specific an empirical treat with the penalty values for best alignment results. • The alignment algorithm is such that with the increase of the penaltyscore produced the increaseof Gaps(caused by deletions and insertions as we remember)and high homology of reminder part of the nucleotide (or other) sequences. However, too big penalties led to the loose of some fraction of nucleotides, which are actually homological, but represented only in some certain sitesof sequences. Our and other authors’experience with mtDNA nucleotidesequences showed that penalties within the limit 15-30 for the gap openingand 0.5-8 for the gap extension are well satisfactory for the first step of the alignment. • WhenCLUSTAL W program have finished [It was runned with the setting in the windows as in our example (Fig. 3.2, А): Gap Opening Penalties («штрафы за открытие пропусков») are 15 units andGap Extension Penalties («штрафы за удлинение пропусков») are 5 units, both for pair-wise and multiple alignment steps], the window appeared that contained the sequences with gaps, looking like blank spaceswith dashes, homologically placed (aligned) sequences (Fig. 3.3). Biggest gaps at this step appearedand sequences looks like as shown in Fig. 3.3.

  14. 3. SEQUENCE ALIGNMENT (6) Fig. 3.3. Window ofCLUSTAL W editor inMEGA, that shows fragments of nucleotide sequencesat Сyt-bgeneafter execution the option “Alignment” («выравнивание»)and realization of the first step of the alignment. Gaps (as blank spaces with dashes) aligned sequences are seen. After gaps removal the sequences take final form as was shown inFig. 3.1, В. The sequences are inspected and large gaps removed manually. One can remove gaps by mean of an editor (processor) software. After first step againCLUSTAL Wdialog box is run and align starts with decreased values of penalties (Fig. 3.2, В). Now after finishing the program all gaps are removed and the obtained file in an appropriate format for further examination.

  15. 4. FINDING AN OPTIMAL MODEL OF NUCLEOTIDE SUBSTITUTION(1) • For choosing a model that is most suitable for particular empirical data sets you need some tool.TheMODELTEST 3.06(Posada, Grandal, 1998) program and later versions 3.6 - 3.7 are very convenient for that. I could not present here info about models but you can easily know on model properties in the program manual and in the literature (Nei, Kumar, 2000; Hall, 2001; Sanderson, Shaffer, 2003; Felsenstein, 2004); there is also a brief info in my book (Kartavtsev, 2005). • To useMODELTESTyou have to learn firstly thePAUP PP, because this program usessome ofPAUP modules. The work with the program is basically simple and includes 5 steps.

  16. 4.FINDING AN OPTIMAL MODEL OF NUCLEOTIDE SUBSTITUTION (2) 1. First you must make a working file in theNexus (.nex) format with the nucleotide sequences and necessary identifiers of the program parameters, in acordance with thePAUP demands; 2. Next you should reach theMODELTEST website and load all recommended modules and copy in the nexus-file made beforethe file“modelblockPAUPb10.txt”, which is distributed with the MODELTEST (it suits forPAUP 4b10 version forWindows); 3. Run then PAUP 4b10installed before (better to renameoriginal data file) and start the executionof the working file; 4. When program stops normally, in the same directory (folder),from which working file have been executed,the new file will appeared with the name“model.scores”; 5. Now it is necessary to run the program, MODELTEST 3.7 is best, froman OSDOS window;better to do this from the directory that contain executable file “modeltest3.7.win.exe”. Consequent identifiers in the command line will beas follows: “modeltest3.7.exe <model.scores> test.out” (last output file may have an arbitrary name). In the output file all necessary information will be presentedand the parameters of one or two best fit modelsof 57 estimated model typeswill be given as well;testing is performed by the Maximum Likelihood (ML) algorithm and by the Acaike Information Criteria.

  17. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (1) • Options and model parameters as well model themselves for calculation of molecular phylogenetic tressare provided by different programs: PAUP* (Swofford, 2000), MEGA-3 (MEGA-4) (Kumar et al., 1993; 2000) etc. Book by Hall(2001) is very good manual for a molecular phylogenetic analysis. This manual is focused mainly onPAUP*.However, in the book the exact examples available and recommendations are given onPP CLUSTAL X, MrBayesetc. • Beginning an analytical jobin MEGA-3 andMEGA-4 may be accomplishedright afteralignment completed. Closing saved file in the Alignment Explorer(редактора выравнивания; it has the extension.mas). Under this action a window appearwith a notice: “Save data to MEGA file: Yes, No, Cancel’ («сохранить файл для MEGA», с опциями: «да», «нет», «сброс»). Choosing the option “YES”opens the next windowwith the file name ready to be saved on the hard disk. By default the file name is supposed same as the alignment file, but with different extension: “.meg”. By choosing the option save(«сохранить»), we run the MEGA PP itself. Before openning themeg-filefor the execution,it is necessary to note in the opened window, what sequence is processed: “Protein-coding nucleotide sequence data” («данные с белок-кодирующей нуклеотидной последовательностью»), withthe alternative YES or NO. At last the dialog box appeared with the question: “ Open Data File in MEGA(«открыть файл с данными в MEGA»), YES, NO.In a choose YESwe getMEGA working file, following by opening a special editor“Sequence Data Explorer” («редактора последовательностей») (Fig. 5.1).

  18. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (2) Fig. 5.1. View of working file inMEGA-3 (MEGA-4) with opened Sequence Data Explorer («редактором последовательностей»). Dots are similar nucleotides. Undefined denoted byR,T,M,W.

  19. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (3) • Close Sequence Data Explorer we have main menu ofMEGA. Main menu ofMEGA contains the following options: File («файл»),Data («данные»), Distances («расстояния»), Phylogeny («филогения»),Pattern («тип»), Selection «отбор»), Alignment («выравнивание»). Option Alignment was considered before (see 5.3). There are two more options in main menu (Windows, Help), which functions are obvious. • Main menu starts with theFile option, which allow several operations with file (Fig. 5.2). Fig. 5.2. Opened window of main menu ofMEGA-3 (MEGA-4) with its options. Opened the dialog box for theFile options with some functions. Command line below gives location of working file (Data File) at the diska tasktitle (Title).

  20. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (4) Fig. 5.4. Opened window of main menu of MEGA-3 (MEGA-4) with its options. A dialog box is opned for the Distances («расстояния»)optionwith several functions. • Distances  Chose Model («выбрать модель»),  Pattern among Lineages («тип между линиями»; 1. Same (Homogeneous) («одинаковые») or (Different (Heterogeneous)(«различные» ). 2. Rates Among Sites («скорость между сайтами»). • To choose an appropriate model allowed the option“Phylogeny”.

  21. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (5) • Next option in main menu isPhylogeny («филогения») (Fig. 5.5). Actions:Construct Phylogeny («построить филогению»), orBootstrap Test of Phylogeny(«бутстреп тест филогении»);give the access to 4 different programs of tree building. • From up to bottom that are: (1) Neighbor Joining; NJ («ближайшего соседства»), (2) Minimal Evolution («минимальной эволюции»), (3) Maximum Parsimony («максимальной парсимонии») and (4) UPGMA(НПГМА). • Comments.

  22. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (6) Fig. 5.5. Opened window of main menu of MEGA-3 (MEGA-4) with its options. The dialog boxof Phylogeny («филогения») andBootstrap Test of Phylogeny («бутстреп тест филогении») are opened; submenu shows main trees allowed to build: (1) Neighbor Joining; NJ («ближайшего соседства»), (2) Minimal Evolution («минимальной эволюции»), (3) Maximum Parsimony («максимальной парсимонии») and (4) UPGMA(НПГМ).

  23. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (7) • Tree building:Bootstrap Test of Phylogeny  Neighbor Joining  Analysis Preferences  Phylogeny Test of Evolution (OptionsBootstrap, Replications= 1000 и Random Seed = 20044 (random number), Model (К2Р, Fig. 5.6). Run optionCompute («вычислить»). We will have tree in theTreeExplorer («исследователя деревьев») (Fig. 5.7). Fig. 5.6. Opened window of main menu of MEGA-3 (MEGA-4) with its options. The dialog boxcontain: Bootstrap Test of PhylogenyNeighbor JoiningPhylogeny Test of Evolution.

  24. 5.TREE BUILDING WITH SOFTWARE PACKAGEMEGA-3 (MEGA-4) (8) Fig. 5.7. TreeExplorer(«исследователь деревьев») ofMEGA-3 (MEGA-4) NJ-tree file opened. Drosophila are on the tips of branches. Tree built on nucleotide sequences of Mdhgene, MEGA (Examples). Branch length is in the bottom. Numbers in the nodes are bootstrap support levels (%).

  25. 6. ANNOTATION ONPAUP, MRBAYESAND SOME OTHER PROGRAMS • Other widely used PP arePAUP 4.0, MrBayes, PHYLIP etc. • PAUP 4.0 (Swofford, 2002): Macintosh («Макинтош»). This PAUP 4.0 version explained in Hall (2001; 2003). For OSWindows there is PAUP 4.0 10b.PAUP 4.0 is very important tool (MODELTEST!). Main its PP: Maximum Likelihood, ML, NJ- and MP Trees. Sustainability of tree quality is finein PAUP. Time in ML is bad property of PAUP; 67 seqat Cyt-b (Kartavtsev et al., 2007a), took 3 weeks. There is PAUPforLinux/Unix. • MrBayes (Hulsenbeck, Rondquist, 2001; Ronquist, Huelsenbeck, 2003) is relatively small PP. Very effective. Set of 67 seq was processed during 2 days. Bayesian trees are MCMC based trees.MrBayesprovides other opportunities, say phylogenetic trees based on morphology. MrBayesis not able to drow a tree. PP TreeView (Page, 1996) is necessary to view a tree and build a consensus tree. • PPPHYLIP (Felsenstein, 1995) is very good tool too. Theoretic background is fine for it (Felsenstein, 2004). PHYLIP gives opportunity to build main trees. Interface is for OSDOS not very convenient.

  26. THANKS!

  27. Ingroup: Внутренние группы Sister group Sister group Ветви Узлы, События видообразования Внутренние узлы Корень Few Terms Сестринские группы Terminal taxa:A B C D E F G HOutgroup: Внешняя Конечные таксоны группа

  28. Dichotomy and Polychotomy Дихотомия и полихотомия Polytomy and Multifurcations Политомия или мультифуркации Bifurcation Бифуркация A A A B C E E C C B D B E D D Unresolved or Star-like Topology Неразрешенная или звездчатая топология Partly Unresolved Topology Частично Неразрешенная топология Fully Resolved Bifurcation Tree Полностью Разрешенное Бифуркационное древо

  29. Unrooted Tree Неукорененное древоThere is no a Possibility to talk on the Direction of Change or on a Descendant Отсутствует возможностьговорить о направленности или о предках на основе такого дерева. Chimp Шимпанзе Cabbage Капуста Monkey Мартышка Fly Муха Rice Рис

  30. Rooted Tree Укорененное древо Human Monkey Human Mosquito Rice Spinach Spinach Mosquito If Rooted Here Если укоренить здесь Rice Root Корень • On Rooted Tree one Could Suggest a Parent-and-Descendant Relationships По укорененному древу можно говорить об отношениях предок - потомок. • Exact Estimate of a Common Hypothetic ancestor Depends on the Place of Rooting Точная оценка общего гипотетического предка зависит От места, куда установлен корень. Monkey

  31. SpeciesB SpeciesC SpeciesA c a b SpeciesA a SpeciesB b SpeciesC Species Tree Видовое древо c Gene Tree Генное древо Difference between the Species Tree and Gene Tree: Duplication of Gene Case

  32. Shortly after speciation, the sister taxa are highly likely to exibit a polyphyletic gene-tree status Вскоре после видообразо- вания сестринские таксоны с высокой вероятностью будут обнаруживать поли-филетический статус генного древа After about 4N generation sister taxa appear reciprocally monophyletic with high probability После 4N поколений сес- тринские таксоны окажутся с высокой вероятностью реципрокно монофилетич- ными Reproductive Isolation Репродуктивная изоляция

  33. Sequence Submission to the GenBankПодписка последовательностей в GenBank (NCBI)

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