Transcript Fig. 1. MtDNA structural map in Torrent catfish, Liobagrus

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: [email protected]
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 package MEGA-3
(MEGA-4).
6. Annotation on PAUP, MrBayes and some other
programs.
APPLICABILITY OF DIFFERENT DNA TYPES IN
PHYLOGENETICS AND TAXONOMY
Species
Genus
Family Order
Spacers
[ITS-1, 2]
mtDNA
nDNA,
rDNA
Most substantiated statistically results
Statistically significant results
Class
Phylum
МАТЕРИАЛ И МЕТОДЫ
1.
DNA Isolation
4.
Phylogenetic
Analysis
2.
PCR DNA
Amplification
3.
Determination of Primary
Nucleotide Sequence
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 or MEGA-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/en or
http://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.
1. SEQUENCE EDITION AND THEIR REGISTRATION IN
THE GENBANK , NCBI (2)
Main task that CROMAS can perform is a comparison of sequences, a removal
of vector sequences in the beginning and in the end of chains, an inversion of the
anti-parallel sequence (chains), a creation of a consensus sequence and 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 representation of 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 sequences obtained with different
primers (1K_F2 etc, left) and they are shown as peaks and their letter translation. After the inversion of the anti-parallel chains
(1KR1_L_p and 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 color for better visual
perception.
1. SEQUENCE EDITION AND THEIR REGISTRATION IN
THE GENBANK , NCBI (3)
After an edition in CHROMAS or any other editor a sequence of nucleotides have to
register it in a gene bank. For a registration of single genes or their segments the
Bankit 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 the
GenBank window.
Data are shown for the
complete mtDNA genome
of one flatfish species
(Pleuronectiformes).
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 formats GenBank,
FASTA etc. In the first case it is looks like as below (Fig. 2.1).
1 gtgcctgagc cggaatagtc ggggacaggc ctaagtctgc tcattcgagc agagctaagc
61 caacctgggt gctctcctgg gagacgacca aatttataac gtaatcgtca ccgcacacgc
121 ctttgtaata atcttcttta tagtaatacc aattatgatn cggagggttc ggaaactgac
181 ttattccatt aataattggg gcccccgnat atggccttcc ctcgaataaa taacatgagt
241 ttctgacttc tacccccatc ctttctcctc cttctagcct cttcaggncg tcgaagctgg
301 ggcagggaca ggatgaaccg tgtatccccc actagctgga aatctagcac acgccggagc
361 atcggtagac ctcaccattt tctctcttca ccttgccgga atttcatcaa ttctaggggc
421 aatcaacttt attactacta tcatcaacat gaaaccaaca gcagtcacta tgtaccaaat
481 cccactattt gtctgagccg tactaatcac cgcacgtcct tcttcttctt tcacactacc
541 acgtcactgg ccgctggcat tacaatgcta ctgactagac cgcaacacta aacacaaaca
601 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
10 with 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 in the 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.
3. SEQUENCE ALIGNMENT (1)
Sequence alignment (выравнивание) is very important procedure, which anticipates their quantitative
analysis including a calculation of similarity-distances measures, homology estimate, and at last building
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 sequence
alignment that make CLUSTAL W, a program adopted for OS Windows.
For the alignment you should first load the sequences into the editor. There are 3 way to do this: (1) Making a
direct record of nucleotide sequences one by one in a consequent window of the editor, (2) Importing the
sequences from a file that 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 with MEGA; cases before (А) and after (В) alignment.
А
3. SEQUENCE ALIGNMENT (2)
В
Fig. 3.1. Windows of the CLUSTAL W alignment editor (Alignment explorer) in
MEGA, with fragments of Сyt-b gene nucleotide sequences from several fish
species before (А) and after alignment completed (В).
With same color similar sites are shown. An asterisk marks 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.
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 unequal and their starts &
ends are 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.
3. SEQUENCE ALIGNMENT (4)
Technically, to start CLUSTAL W execution you have to choose all 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) under pair-wise alignment (Pairwise Parameters) and multiple alignment (Multiple
Parameters).
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 penalty score
produced the increase of 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 sites of sequences. Our and other authors’ experience with mtDNA
nucleotide sequences showed that penalties within the limit 15-30 for the gap
opening and 0.5-8 for the gap extension are well satisfactory for the first step of
the alignment.
When CLUSTAL 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 and Gap 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 spaces with dashes, homologically placed (aligned)
sequences (Fig. 3.3). Biggest gaps at this step appeared and sequences looks
like as shown in Fig. 3.3.
3. SEQUENCE ALIGNMENT (6)
Fig. 3.3. Window of CLUSTAL W editor in MEGA, that shows fragments of nucleotide
sequences at Сyt-b gene after 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 in Fig. 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 again CLUSTAL W dialog 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.
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. The
MODELTEST 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 use MODELTEST you have to learn firstly the PAUP
PP, because this program uses some of PAUP modules.
The work with the program is basically simple and
includes 5 steps.
4. FINDING AN OPTIMAL MODEL OF NUCLEOTIDE
SUBSTITUTION (2)
1. First you must make a working file in the Nexus (.nex) format with the nucleotide
sequences and necessary identifiers of the program parameters, in acordance
with the PAUP demands;
2. Next you should reach the MODELTEST website and load all recommended
modules and copy in the nexus-file made before the file
“modelblockPAUPb10.txt”, which is distributed with the MODELTEST (it suits for
PAUP 4b10 version for Windows);
3. Run then PAUP 4b10 installed before (better to rename original data file) and start
the execution of 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, from an OS
DOS window; better to do this from the directory that contain executable file
“modeltest3.7.win.exe”. Consequent identifiers in the command line will be as
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 presented and the parameters of
one or two best fit models of 57 estimated model types will be given as well;
testing is performed by the Maximum Likelihood (ML) algorithm and by the Acaike
Information Criteria.
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (1)
Options and model parameters as well model themselves for calculation of
molecular phylogenetic tress are 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 on PAUP*. However, in the book the exact examples available and
recommendations are given on PP CLUSTAL X, MrBayes etc.
Beginning an analytical job in MEGA-3 and MEGA-4 may be accomplished right after
alignment completed. Closing saved file in the Alignment Explorer (редактора
выравнивания; it has the extension .mas). Under this action a window appear with 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 window with 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 the meg-file for 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»), with
the 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
YES we get MEGA working file, following by opening a special editor “Sequence
Data Explorer” («редактора последовательностей») (Fig. 5.1).
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (2)
Fig. 5.1. View of working file in MEGA-3 (MEGA-4) with opened Sequence
Data Explorer («редактором последовательностей»).
Dots are similar nucleotides. Undefined denoted by R,T,M,W.
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (3)
Close Sequence Data Explorer we have main menu of MEGA. Main menu of MEGA 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 the File option, which allow several operations with file (Fig. 5.2).
Fig. 5.2. Opened window of main menu of MEGA-3 (MEGA-4) with its options.
Opened the dialog box for the File options with some functions. Command line below gives location
of working file (Data File) at the disk a task title (Title).
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-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») option with 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”.
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (5)
Next option in main menu is Phylogeny («филогения»)
(Fig. 5.5). Actions: Construct Phylogeny («построить
филогению»), or Bootstrap 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.
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (6)
Fig. 5.5. Opened window of main menu of MEGA-3 (MEGA-4) with its options.
The dialog box of Phylogeny («филогения») and Bootstrap 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).
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (7)
Tree building: Bootstrap Test of Phylogeny  Neighbor Joining  Analysis Preferences
 Phylogeny Test of Evolution (Options Bootstrap, Replications = 1000 и Random Seed
= 20044 (random number), Model (К2Р, Fig. 5.6). Run option Compute («вычислить»). We
will have tree in the TreeExplorer («исследователя деревьев») (Fig. 5.7).
Fig. 5.6. Opened window of main menu of MEGA-3 (MEGA-4) with its options.
The dialog box contain: Bootstrap Test of Phylogeny  Neighbor Joining 
Phylogeny Test of Evolution.
5. TREE BUILDING WITH SOFTWARE
PACKAGE MEGA-3 (MEGA-4) (8)
Fig. 5.7. TreeExplorer
(«исследователь
деревьев») of MEGA-3
(MEGA-4)
NJ-tree file opened.
Drosophila are on the tips
of branches. Tree built on
nucleotide sequences of
Mdh gene, MEGA
(Examples). Branch length
is in the bottom. Numbers
in the nodes are bootstrap
support levels (%).
6. ANNOTATION ON PAUP, MRBAYES AND
SOME OTHER PROGRAMS
Other widely used PP are PAUP 4.0, MrBayes, PHYLIP etc.
PAUP 4.0 (Swofford, 2002): Macintosh («Макинтош»). This PAUP 4.0
version explained in Hall (2001; 2003). For OS Windows 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 fine in
PAUP. Time in ML is bad property of PAUP; 67 seq at Cyt-b (Kartavtsev et
al., 2007a), took 3 weeks. There is PAUP for Linux/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. MrBayes provides other
opportunities, say phylogenetic trees based on morphology. MrBayes is not
able to drow a tree. PP TreeView (Page, 1996) is necessary to view a tree
and build a consensus tree.
PP PHYLIP (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 OS DOS not very convenient.
THANKS!
Few Terms
Ingroup: Внутренние группы
Sister group
Terminal taxa: A
B
C
Сестринские
группы
D
E
Sister group
F
G
H Outgroup: Внешняя
Конечные таксоны
группа
Ветви
Узлы,
События видообразования
Внутренние узлы
Корень
Dichotomy and Polychotomy
Дихотомия и полихотомия
Polytomy and Multifurcations
Политомия или мультифуркации
Bifurcation
Бифуркация
A
A
B
C
C
E
A
E
C
D
B
B
E
Unresolved or
Star-like Topology
Неразрешенная
или звездчатая
топология
D
Partly Unresolved
Topology
Частично
Неразрешенная
топология
D
Fully Resolved
Bifurcation Tree
Полностью
Разрешенное
Бифуркационное
древо
Unrooted Tree
Неукорененное древо
There is no a Possibility to talk on the Direction of Change or
on a Descendant
Отсутствует возможность говорить о направленности или
о предках на основе такого дерева.
Chimp
Шимпанзе
Cabbage
Капуста
Monkey
Мартышка
Rice
Рис
Fly
Муха
Rooted Tree
Укорененное древо
 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.
Human
Monkey
Human
Mosquito Rice Spinach
Spinach
Monkey
Rice
If Rooted Here
Если укоренить
здесь
Mosquito
Root
Корень
Difference between the Species Tree and Gen
e Tree: Duplication of Gene Case
Species A Species B
a
b
Species C
c
Species A
a
Species B
b
Species C
Species Tree
c
Видовое древо
Gene Tree
Генное древо
Shortly after speciation, the s
ister taxa are highly likely to
exibit a polyphyletic gene-tre
e status
Вскоре после видообразования сестринские таксоны
с высокой вероятностью
будут обнаруживать поли-ф
илетический статус
генного древа
Reproductive Isolation
Репродуктивная изоляция
After about 4N generation sist
er taxa appear reciprocally m
onophyletic with high probabili
ty
После 4N поколений сестринские таксоны окажутся
с высокой вероятностью
реципрокно монофилетичными
Sequence Submission to the GenBank
Подписка последовательностей в GenBank (NCBI)
What are Barcodes?
Barcodes are short nucleotide sequences from a standard genetic locus for use in species identification. Currently,
the Barcode sequence being accepted for animals is a 5' 650 base pair region of the mitochondrial cytochrome
oxidase subunit I (COI) gene.
What does the Barcode Submission tool do?
The Barcode Submission tool provides for streamlined online submission of Barcode sequences into GenBank. With
this tool, one can:

submit new Barcode sets

complete your most recent incomplete submission

download a flat file summary of completed submissions
How does the Barcode Submission tool work with My
NCBI?
My NCBI is a central place to customize NCBI Web services. The Barcode Submission tool associates your
Barcode submissions with your My NCBI user name and remembers your contact information to expedite future
Barcode submissions. Barcode also associates your most recent incomplete submission with your My NCBI
username so that if you're interrupted while submitting a Barcode set, you can complete the submission later.
To register for My NCBI, follow the link at the bottom of this page to Sign in to Use Barcode Submission Tool and
click register for an account on the My NCBI Sign In page. Read My NCBI Help for more information about My
NCBI.
In order to ensure that the My NCBI user currently using the Barcode Submission tool is the person submitting the
Barcode set, you will be prompted for your My NCBI user name and password before you begin a Barcode
submission.
What is needed to submit a Barcode set?

A My NCBI Account (register on My NCBI Sign In page)

A web browser that supports both JavaScript and cookies

The title of a published or in-press paper that discusses the Barcode Set

A text file of the set of nucleotide sequences in FASTA format

The names or sequences of forward and reverse primers

A tab-delimited table of source modifier data for the set

A text file of the set of protein sequences in FASTA format (optional)

A tab-delimited table of trace attributes and a compressed archive containing the traces (optional)