Bioinformatics complete manual

Manual of Bioinformatics
Submitted to:
Muhammad Ibrahim
Submitted By:
Muhammad Zeeshan Ahmed
Roll No:
BS-BC-14-17
BS Biochemistry
7th Semester
Session 2014-2018
Department of Biochemistry,
Bahhauddin Zakariya University (BZU),
Multan.

Table of Content
Sr.# Experiment
1 How to search a paper?
2 Downloading of sequence and paper from NCBI (FASTA+BLAST)
3 Inter-conversion of protein and amino acids and Perform BLAST on it.
4 Construction of phylogenetic tree
5 Use of phylogeny free software for phylogenetic tree analysis
6 How to download mega -7 ( Molecular Evolutionary Genetic Analysis)
7 CURATION USING MEGA 7 SOFTWARE
8 PRIMER DESIGNING TOOLS
9 Global sequence alignment
10 Primer quest
11 HOW TO USE SEQUENCE CLEANER?
12 HOW TO DOWNLOAD SEQUIN SOFTWARE?
13 Amino Acid Explorer
14 How to work with ExPASY?
15 DNA/RNA Cleaner and Sequence Cutter
16 DNA/RNA Translator
17 DNA/RNA NetPlasmid
18 ORF viewer
19 Batch codon usage
20 Aligner
21 Complementor
22 EnzFinder
23 OligoCalc
24 Primer3
25 PrimerAnneal
26 Protein Cleaner and ProtCalc
27 Sequence Aligner
28 PatSearch
29 Plot Analysis
30 SeqPainter
31 Arrays
32 Array Average Calculator
33 Array Organizer
34 Array List Labelizer
35 Cell Bio SMS2 Tool
36 Curation of Phylogeny Free

Experiment No 1. DATE: 17-08-17
How to download of protein or gene or nucleotide sequence from NCBI and
Format conversion?
 Write www.ncbi.nlm.nih.gov/ on Google chrome
 National Center for Biotechnology Information will open
 Select gene/protein from “all data base’

 Write the name of protein/gene on the box and click on Search.
 All paper related to topic will appear click on desire paper, complete paper will open
 Copy the sequence given on the last of page in the heading of ORIGIN by pressing Ctrl+ C.
 Paste this sequence on new word page.
 We can change the format of sequence by clicking on FASTA on the top of page.

EXPERIMENT NO. 2 DATE: 17-08-17
Searching paper from NCBI (National Centre for Biotechnology and Information)
 Type NCBI in Google search bar.
 Click on www.ncbi.nlm.nih.gov.
 Open it and go to above heading All Database.
 Write name of protein/gene in next bar.
 Then click on search button.
 List of different organisms will be opened regarding that protein/gene.
 Click on protein of desired organism (complete paper of that organism will appear).
 All the papers related to topic will appear then click on desire paper, complete paper will open.

How sequences are blast?
 Copy FASTA format sequence and reopen NCBI website in a new tab.
 Click on Basic Local Alignment Search Tool (BLAST)
 Click on Nucleotide Blast or Protein Blast according to your sequence.
Web BLAST
 A page open. Paste the sequence in box.
 Click on Blast
 Graphic Summary, Descriptions, Alignments of the nucleotide sequence will appear.
Another method of Blast
 Another method of BLAST is also available in NCBI website.
 Open NCBI website and open required paper just like above method.
 Copy sequence present at the bottom of paper and select Run Blast option present at right side
of that paper. Ultimately sequence will be Blast and different properties (like Alignment of
nucleotide sequence, Graphic summary and description) will appear.

Conversion of protein into gene/nucleotide sequence
 Protein sequence back-translation is used to predict the possible nucleic acid sequence from a
specified peptide sequence
 In new tab write sequence translator tool.
 Click on Bioinformatics Tools for Sequence Translation < EMBL-EBI
 www.ebi.ac.uk/Tools/st
 From protein sequence back- translation Click on Launch Backtranseq.
 Paste the sequence in box. And Click on SUBMIT.
 Protein sequence is converted into Gene sequence.
 Now sequences are ready to do BLAST.
Conversion of gene/nucleotide sequence into protein
 Nucleotide sequence translation: is used to translate nucleic acid sequence to corresponding
peptide sequences.
 Click on First option “Launch transeq” for nucleotides to convert into protein.
 Paste the copied sequence (FASTA formatted) in box.
 Click on submit button.
 Protein sequence will appear in few seconds with symbol “>”.
 Now that sequences are ready to do BLAST.

How to Download MEGA 7 (Molecular Evolutionary Genetic Analysis)?
 Open Google chrome.
 Then in new tab write down MEGA-7.
 Click on Official site.
 Mega: Official site.
 www.megasoftware.net/
 Click on “Download”.


 Fill the information requested below e.g click on student and university and write down your
university name.
 Click on “Download file” info will open.
 Click on “Start Download”.
 When download complete click on CLOSE.
 Mega-7 software has been downloaded.
INSTALLATION OF MEGA-7 IN YOUR COMPUTER:
 Open Mega-7 from your computer from downloads.
 “User Account Control” tab will appear then click on YES (allow software to make changes
on your computer).
 Then in select language setup select ENGLISH.
 Click on OK.
 Then click on NEXT continuously 3 times.
 Select Additional tasks by clicking on Create Desktop Icon.
 Click on INSTALL and then on FINISH.
 Setup of MEGA-7 will be installed in your computer.

Phylogenetic Tree Using MEGA 7/Offline
 Open the file and select the sequences of proteins of different organisms online or the sequence
you have already saved.
 ii) select using control +A, iii) copy using control + C.
 Open Mega 7.
 Open alignment.
 Select Edit/ Build Alignment.
 Select create a new alignment and OK.
 Select the option of proteins because we are using proteins not DNA
 A new window will open that will show protein sequence. Select the lower small box and the
press control + N. Then again press control + V.
 Sequences will appear
 Delete the sequence No.1.
 After deleting sequences will appear in colored form.
 Open data icon.
 Select phylogenetic analysis.
 Minimize this window.
 Open phylogeny.
 Select the option construct/ Maximum likelihood tree.
 Yes the option.
 OK the compute option and wait few seconds phylogenetic tree after the completion of % will
appear with respect to proteins sequences, similarly you can construct the tree using DNA
sequences.
 After appearance of tree all the information about tree will be displayed below.

CURATION AND CONSTRUCTION OF PHYLOGENETIC TREE USING
MEGA 7 SOFTWARE/TOOL
 Select your sequences in the Fasta Format (you can also change the sequence name if needed
but the sign of greater than remains as such)
 Copy these sequences by pressing control+C
 Open Mega 7 software/tool
 Click on align
 Click on Edit/Build Alignment a box will appear
 Click on create a new alignment
 Click on OK box
 Click on DNA then a new window will open
DNA Sequences Translated Protein Sequences
 Click on right sided small blue box.
 Click on Control+N
 Click on Control+V

 Click on any sequence, all the sequences will appear in different colours.
 Click on the last sequence1 which has no nucleotide sequence and click on delete key to delete
it
 You can rename/change the name of each sequence if you want in this stage, for this purpose
select the space of name and click on backspace then write new name of sequence
 Click on Alignment
 Click on Align by Clustal W
 Select all and click on OK button
 Again click on OK button in the new box the sequences will arrange themself in pairwise
alignment and multiple alignment in few seconds.
 All the sequences will appear in red colour in the form of maximum matching, mismatching
and gaps
 Click on any one sequence, then all the sequences will appear in different colours
 Drag the small box which normally appear on the right side towards left side and do curation
by deleting maximum sequences from start and at the end of your sequences due to maximum
gaps, and delete the gap from middle if any is present by clicking on column on the top, it will
select automatically then click on delete key to delete it. Do maximum curation and try to
delete all the gaps for best tree construction. Best curation gives best phylogenetic tree.
Note:
 It should be noted that stars show 100% matches
 After curation save this file. for this purpose click on Data
 Click on save session
 Name the file and select its destination e.g desktop, D, E or F drive etc.
 Click on save button
 Open the Mega 7 and click on phylogeny
 Click on Construct/ Test Neighbour-Joining Tree for DNA and Construct /Test Maximum
likelihood for proteins. You can construct protein tree for it the process is same but you have
to select proteins instead of DNA
 Pick your file from Mega file.
 Go to all files find the destination/drive of your saved file of sequences and click on it
 Click on compute and then the phylogenetic tree will appear in few seconds. You can save
your phylogenetic tree for your result
 Modify its shapes according to your choice.
 Percentage given below the tree shows validity of your tree.
 Percentage must be in between 0.0 something like that.

PHYLOGENY FREE/Online
 Open the Google tab. Type phylogeny free in search bar.
 Open the first link that is Phylogeny.fr: Home(www.phylogeny.fr)
 A phylogeny free page will appear.
 Drag the arrow on Phylogeny Analysis.
 Three options will appear. Select “ONE CLICK”
 Copy the sequence of gene or protein in FASTA format.
 Paste the sequence in the given block.
 Submit the sequence. Wait for a few seconds.
 A phylogeny tree will appear.
 Now modify it according to your requirements.

Primer quest
 Write primer quest in Google search bar.
 Choose the link: Primer Quest Tool | IDT - Integrated DNA Technologies
 Now enter the sequence.
 Paste your sequence into the Textbox
 Add up to 50 sequences in FASTA format
 Sequence length must be greater than 80 bases
 Primer Quest accepts only nucleic acid bases
 Click on 1st block and wait for a while, the result page will appear,
 It shows 5 sets of gene sequence choose highest value amplicon length like this as given below:
 For detail click on View Assay Details.
 Then click on 2nd block it shows the probe as well,
How to Download Sequin Software?
 Write Download Sequin in Google box.
 Click on the link Download Sequin-NCBI.
 A new page will open.
 Click on a blue link showing “Click here for instructions on how to download using FTP”.
 A new page will open.
 In this page, different links will be given to download sequin for different types of systems e.g
windows, Mac OS X, Linux etc.
 Downloading window will open for sequin download.
 Hence, Sequin is downloaded.

Experiment 12
Submission Sequence into the GenBank through Sequin.
 Open the sequin and click on the new submission.
 The pages in the Submitting Authors form ask you to provide the release date, a working title,
names and contact information of submitting authors, and affiliation information. To create a
personal template for use in future submissions, use the File/Export option after completing
each page of this form.

 The Contact page asks for the name, phone number, and e-mail address of the person
responsible for making the submission. Database staff members will contact this person if
there are any questions about the record.
 In the Authors page, enter the names of the people who should get scientific credit for the
sequence presented in this record. These will become the authors for the initial (unpublished)
manuscript.

 The Affiliation page asks for the institutional affiliation of the primary author.
 The traditional submission is of a single nucleotide sequence containing one or more genes
and encoding one or more proteins. Data files should be prepared in FASTA
format. Segmented sequences, e.g., where several exons have been sequenced but the
complete introns are not yet sequenced, may also be submitted, with the individual nucleotide
segments in FASTA format combined into one file.

 Organism information is most easily entered by selecting the appropriate organism from the
scrollable list. As you begin typing the scientific name, the list will jump to the right
alphabetical location. Click on the list to finish the selection.
 To import the nucleotide sequence data, click on the Nucleotide folder tab or the Next
Page button to advance to the next page. Select molecule type and topology, check any
additional boxes that apply, then click on Import Nucleotide FASTA and select the appropriate
file.

 After you have completed importing the data files, Sequin will display your full submission
information in the GenBank format (or EMBL format if you chose EMBL as the database for
submission in the first form).

How sequences clean in just bio
 First of all type Just bio in Google box.
 Open first link as Hosted Tools | JustBio - sequence sequences (www.justbio.com).
 A page will open. At the left side of paper log in option will present. Give user name
and password in it. And click log in option.
 Same page open with logged account means that you have access to use just bio
software.
 Then Click at www.Cellbiol.com Resources for Molecular and Cell Biologists.
 A page open contains that options, click sequence cleaner
 Then another bar open, just like previous one.
 This application supports degenerated/ambiguous IUPAC characters. Numbering may
change 10, 15, 20, 50 etc.
 Now put your DNA/Protein sequence in box and press clean it

Amino Acid Explorer
 Open the web page.
 Type NCBI in Google search bar.
 Click on www.ncbi.nlm.nih.gov.
 Then click on Resource List (A-Z).
 A site map will open then click on the Amino Acid Explorer.
 Then different properties of amino acids will appear.
 Now click on the display table on the Biochemical properties.
 All the details related to amino acids will appear in the form of a table e, g 1-letter
code, 3-letter code, full name, its chemistry and abundance (%).
 Click on the Add/Remove data columns.
 Then tick mark all the unmarked properties in that given list.
 Now click on the Draw Table.
 A table will appear which contains all the relevant properties of each amino acid
e,g side chain properties, interaction modes, molecular weight, isoelectric point
and standard codons etc.
 Now we can compare the properties of any two amino acids by clicking on the
COMPARE on the side tool bar.

How to download MedChem?
 Open the Google search bar.
 Write MedChem Designer (free) Download windows version.
 Then click on the START DOWNLOADING.
 After that install that software, click on INSTALL and then FINISH.
 Setup of MedChem now installed.

Use of ExPASY to study the protein expression
 Open the web page.
 Type ExPASY on the Google search bar.
 Now select the ExPASY: SIB Bioinformatics Resource Portal-Home.
 A page will open and the popular resources will appear on the right side of this
page.
 From these resources click on the SWISS-MODEL.
 Then click on START MODELLING.
 Now place your target sequence in the given bar.
 When the colored lines appear then click on BUILD MODEL.
 It will take a little time to build a model of the given sequence. Now rotate the
model and take pictures of the model from different angles.

Tools on JustBio Website
DNA/RNA Tools
1. DNA/RNA Cleaner and Sequence Cutter
 First of all open the Google chrome and type http://www.justbio.com/hosted-tools.html on search
bar.
 Now click the “Cleaner” on the DNA/RNA side

 Now select and copy the sequence from gene library and paste it on the just Bio cleaner and click
on clean it
 Now click on the “Send it to Cutter”, select the specific enzymes and click on “Cut”.
 Enzyme name, recognition seq. and Position of recognition sites are shown down in the end of
restricted fragments.

2. DNA/RNA Translator
 To translate the DNA sequence from 1 to 6 phases, click on the “Translator” on the DNA/RNA
side.
 Then paste the DNA sequence on the translator input form and select the required phase.
 After selecting the phase, click on the “Translate”.
 Now paste the nucleotide sequence, select the I phase, 3 phase or 6 phase as you required.
 And then click on Translate.

3. DNA/RNA NetPlasmid
 This tool is used to draw the coloured plasmid map in a few seconds, click on the NetPlasmid on
the DNA/RNA side.
 Click the “NetPlasmid” down the DNA/RNA side on the “JustBio” home page.
 Now click on the “Automatic draw from DNA sequence”.
 Then copy a DNA sequence from the gene library and paste it on the given space and then select
the enzymes and also the restriction enzymes to be used.
 Now click on “Import and draw”.
 So, the online plasmid map of the inserted sequence will be shown in the form of figure on the
screen.
 You can also update and again draw the net plasmid map of the same sequence.

4. ORF viewer
 An ORF is a sequence of DNA that starts with start codon “ATG” (not always) and ends with any
of the three termination codons (TAA, TAG, TGA).
 With this tool, the ORF regions on any DNA sequence can find out easily. The graphical output on
the 6 phases is obtained (direct and reverse orientations).
 Click on the “ORF Viewer” on the DNA/RNA side on.
 SO, the ORF viewer input form will open.
 Now paste your DNA sequence in the input form.
 Select the Vertebrate Mitochondria in the codon table option.
 Then click on “Find ORFs”.

5. Batch codon usage
 This tool of just bio is used to calculate the %GC and codon usage rates from a list of sequences.
Paste your sequence list in FASTA format.
 First of all open just bio and click on the “Batch Codon-Usage” on the DNA/RNA side.
 Batch Codon-Usage Input Form will open.
 Now select the DNA sequences from your gene library and copy-paste the sequence list in FASTA
format
 Click on YES to extract the ORF.
 Then click on the “START” button.
 A page will open which shows the %GC in ORFs and the Codon-Usage Means in ORFs.

6. Aligner
 This tool is used to align the multiple sequences of DNA or protein, based on the Clustal W engine.
 Click on the “Aligner” on the DNA/RNA side on justBio.
 Aligner input form will open.
 Paste the nucleotide sequence in the first block and give the sequence name.
 Paste another nucleotide sequence in the next block which is too be aligned with the first sequence.
 You can add other sequences by clicking on the “add one sequence +”, repeat the same above steps

 Now submit your request and scroll down to see your aligner Report.
7. Complementor
 This is used to find out the complementary of any sequence.
 Click on the “Complementor” on the DNA/RNA side on the JustBio page.
 So the Complementor input form will open.
 Select the DNA sequence from your gene library and copy-paste that sequence.
 Then click on the “Complement” button.
 Now the complementary of that sequence will appear.

8. EnzFinder
 With this tool you can search for commercial data about any Enzyme, its availability and cut
sequence references etc.
 Open just bio and click on the “EnzFinder” on the DNA/RNA side.
 EnzFinder input form will open.
 Now enter the restriction enzyme name like “EcoR1”or the part of it here.
 Then click on the Search option.
 Now select one of opened list enzyme according to your require.
 The Enzyme Data will appear in the EnzFinder Result.
9. OligoCalc
 This tool of just bio is used to calculate the primer parameters (Tm, %GC).
 Open just bio and click on the “OligoCalc” on the DNA/RNA side.
 Then the OligoCalc Input Form will appear.

 Paste the copied Oligo sequence here from the library.
 Now click on the “calculate” option on the oligocalc input form.
 So the length, %GC, melting temperature and molecular weight is calculated.
10. Primer3
 This tool is used to pick the primers from a DNA sequence and maybe of complex usage.
 Click on the “Primer3” on the DNA/RNA side on JustBio.
 Then a Primer Input Form will open.

11. PrimerAnneal
 With this tool of justbio, you can visualize where your primers will hybridize on your DNA
template sequence.
 Click on the “PrimerAnneal” on the DNA/RNA side on the JustBio.
 A primer anneal detection or search for a sequence motif will open.
 Now fill this motif with the required data.
 Click on the “Search” option. Then the result will appear.

Protein Tools
1. Protein Cleaner and ProtCalc
 With cleaner tool you can simply and quickly clean your sequence from unwanted text, marks,
spaces etc.
 And with the help of “ProtCalc” you can simply and quickly determine statistical, theoretical
molecular weight, isoelectric point and extinction data from your protein sequence.
 Click the “Cleaner” down the protein side on the “JustBio” home page.
 Paste your Amino Acid Sequence, select protein and clean it and send it to the “ProtCalc” and
then click on “Execute”.
 It will calculate the molecular weight of your sequence, Number of each individual amino acid
and total number of amino acids and theoretical pI (isoelectric point).

2. Sequence Aligner
 With this tool you can align your multiple sequences of protein, based on a Clustalw
engine.
 Click the “Aligner” down the protein side on the “JustBio” home page.
 Paste the amino acid sequence in the first block and give the sequence name.
 Paste another amino acid sequence in the next block which is too be aligned with the first sequence.
 You can add other sequences by clicking on the “add one sequence +”, repeat the same above steps
 Now submit your request and scroll down to see your aligner Report.

3. PatSearch
 This tool is used to search the “PROSITE” pattern on your protein sequence where PROSITE is
a protein database. It consists of entries describing the protein families, domains and functional
sites as well as amino acid patterns and profiles in them.
 Click the “PatSearch” down the protein side on the “JustBio” home page.
 Now enter your amino acid sequence in the box and click on the Search.
 The result will show you the PROSITE Pattern and their positions on your sequence.

4. Plot Analysis
 This tool generates hydropathy and Antigenic plots (Kyte-Doolittle and Hopp-Woods) of your
protein sequence.
 Click the “Plot Analysis” down the protein side on the “JustBio” home page.
 Type or paste your Protein sequence in the protein plots input form.
 Kyte and Doolittle Plot (hydropathy) with window size of 9 is selected.
 Then click on Draw Plot.
 The Kyte and Doolittle Plot will be drawn in front of you.

 Now again go back to the protein sequence input form, paste your protein sequence
 Select Hoop and Woods Plot (Hydrophilic/antigenic) with window size of 6.
 And click on draw plot.
 Hoop and Woods Plot (Hydrophilic/antigenic) will be drawn in front of you.
5. SeqPainter
 This tool will color your amino acid sequence according to their properties (Default by
hydrophobicity)
 Click the “SeqPainter” down the protein side on the “JustBio” home page.

 Type or paste your Protein sequence in the SeqPainter input form.
 You can select your amino acids with different colors like Red, Green, Yellow and Blue.
 You can also increase or decrease the Font Size.
 Now Click on Paint sequence and get your protein sequence colored according to residues types.
File Processing and Transposition.
1. Arrays
 Arrays have several tools to batch process and analyze array genomic data. It can be used also for
lists of values to treat.
 Click the “Arrays” down the File Processing and Transposition side on the “JustBio” home page.
 Array Tools will contain “Array Averages calculator, Array Organizer and Array List Labelizer”.
a) Array Average Calculator.
 This tool allows you to batch calculate averages on an unlimited sets of values. This is useful when
doing replicates and compiling the average. You can also use it for whatever list of results you
have, not only on microarrays.
 Click on the “Array Average Calculator” on the Array Tools page.

 Now paste your data set, you can also get guideline from sample file.
 And submit your data.
 The array_avg will show you the result of your data.

b) Array Organizer
 This tool allows you to compare and organize intensities or ratios for the same ORF for several
probes or comparisons.
 Click on the “Array Organizer” on the Array Tools page.
 Now paste your data set, you can also get guideline from sample file.
 The array org will show you the result of your data.
c) Array List Labelizer.
 This tool allows you to associate descriptions and other texts to an ordered list of labels (ex: A ORF
gene list). It keeps the order and associate description from a flat database you provide.
 Click on the “Array List Labelizer” on the Array Tools page.

 Now write order list of labels and Database with format.
 The list label will show you the result of your data.
Sequence Manipulation Suit
Format Conversion
1. Combine FASTA
 Combine FASTA converts multiple FASTA sequence records into a single sequence. Use
Combine FASTA, for example, when you wish to determine the codon usage for a
collection of sequences using a program that accepts a single sequence as input.
 First of all open the Google chrome and type http://www.justbio.com/hosted-tools.html
on search bar.
 Then click on www.cellbiol.com and the BIO-WEB will open.

 Now click on SMS2 and click on the Combine FASTA.
 Then the combine FASTA form will open where the FASTA sequence will be pasted.

 Paste the sequence in the text area and click on submit.
 The result will appear.
2. EMBL to FASTA
 EMBL to FASTA accepts an EMBL file as input and returns the entire DNA sequence in
FASTA format. Use this program when you wish to quickly remove all of the non-DNA
sequence information from an EMBL file.
 In the SMS2, click on the EMBL to FASTA.

 Now paste the one or more EMBL files into the text input form and click on the submit
option.
3. EMBL feature Extractor
 EMBL Feature Extractor accepts an EMBL file as input and reads the sequence feature
information described in the feature table. The program extracts or highlights the relevant
sequence segments and returns each sequence feature in FASTA format. EMBL Feature
Extractor is particularly helpful when you wish to derive the sequence of a cDNA from a
genomic sequence that contains many introns.
 In the SMS2, click on the EMBL feature Extractor.

option.
4. EMBL Trans Extractor
 EMBL Trans Extractor accepts an EMBL file as input and returns each of the protein
translations described in the file in FASTA format. EMBL Trans Extractor can be used
when you are more interested in the predicted protein translations of a DNA sequence
than the DNA sequence itself.
 In the SMS2, click on the EMBL Trans Extractor.

option.
5. Filter DNA
 Filter DNA removes non-DNA characters from text. Use this program when you wish to
remove digits and blank spaces from a sequence to make it suitable for other
applications.
 In the SMS2, click on the Filter DNA.

 Now paste text or the DNA sequence into the text input form and click on the submit
option.
6. Filter Protein
 Filter Protein removes non-protein characters from text. Use this program when you wish
to remove digits and blank spaces from a sequence to make it suitable for other
applications.
 In the SMS2, click on the Filter Protein.

 Now paste the Protein sequence into the text input form and click on the submit option.
GenBank to FASTA
 GenBank to FASTA accepts a GenBank file as input and returns the entire DNA sequence in
sequence information from a GenBank file.
 Search the http://www.justbio.com/hosted-tools.html on the Google chrome search bar and
then click on the www.Cellbiol.com on the right side of the below the log in side.
 You can also search the Cell Bio by entering the http://www.cellbiol.com/ on the Google
chrome search bar.

 Then click on the SMS2 besides the Home.
 Now click on the GenBank to FASTA on the right side down the SMS.
 Paste the contents of one or more GenBank files into the text area and submit it.
 The result will come as shown down.

GenBank Feature Extractor
 GenBank Feature Extractor accepts a GenBank file as input and reads the sequence feature
information described in the feature table, according to the rules outlined in the GenBank release
notes. The program extracts or highlights the relevant sequence segments and returns each
sequence feature in FASTA format. GenBank Feature Extractor is particularly helpful when you
wish to derive the sequence of a cDNA from a genomic sequence that contains many introns.
 Search the http://www.justbio.com/hosted-tools.html on the Google chrome search bar and then
click on the www.Cellbiol.com on the right side of the below the log in side.
 You can also search the Cell Bio by entering the http://www.cellbiol.com/ on the Google chrome
search bar.
 Now click on the GenBank Feature Extractor on the right side down the SMS.

GenBank Trans Extractor
 GenBank Trans Extractor accepts a GenBank file as input and returns each of the protein
translations described in the file in FASTA format. GenBank Trans Extractor should be used when
you are more interested in the predicted protein translations of a DNA sequence than the DNA
sequence itself.
search bar.
 Now click on the GenBank Trans Extractor on the right side down the SMS.

One to Three
 One to Three converts single letter translations to three letter translations.
chrome search bar.
 Now click on the One to Three on the right side down the SMS.

Range Extractor DNA
 Range Extractor DNA accepts a DNA sequence along with a set of positions or ranges. The bases
corresponding to the positions or ranges are returned, either as a single new sequence, a set of
FASTA records, as uppercase text, or as lowercase text. Use Range Extractor DNA to obtain
subsequences using position information.
search bar.
 Now click on the Range Extractor DNA on the right side down the SMS.
 Paste a raw sequence or one or more FASTA sequences into the text area.
 Now enter the base positions or ranges to be extracted. Use ".." to represent a range, and use a
comma to separate entries. The words 'start', 'end', 'center', and 'length' can be used in place of
digits, to represent the beginning, end, middle, and length of the sequence. Arithmetic expressions
can be included in the ranges. For example, to obtain the last three bases of a sequence, the range
'(end - 2)..end' can be used. To obtain the 30 bases on either side of the center base along with the
center base, the ranges '(center - 30)..(center - 1), center, (center + 1)..(center + 30)' can be used
and submit it.
Range Extractor Protein

 Range Extractor Protein accepts a protein sequence along with a set of positions or ranges. The
residues corresponding to the positions or ranges are returned, either as a single new sequence, a
set of FASTA records, as uppercase text, or as lowercase text. Use Range Extractor Protein to
obtain subsequences using position information.
search bar.
 Enter the residue positions or ranges to be extracted. Use "...” to represent a range, and use a
can be included in the ranges. For example, to obtain the last three residues of a sequence, the
range '(end - 2)..End' can be used. To obtain the 30 bases on either side of the center residue along
with the center residue, the ranges '(center - 30)..(center - 1), center, (center + 1)..(center + 30)'
can be used and submit it.

GenBank to FASTA
 GenBank to FASTA accepts a GenBank file as input and returns the entire DNA sequence in
sequence information from a GenBank file.
chrome search bar.
 Now click on the GenBank to FASTA on the right side down the SMS.

GenBank Feature Extractor
 GenBank Feature Extractor accepts a GenBank file as input and reads the sequence feature
information described in the feature table, according to the rules outlined in the GenBank release
notes. The program extracts or highlights the relevant sequence segments and returns each
sequence feature in FASTA format. GenBank Feature Extractor is particularly helpful when you
wish to derive the sequence of a cDNA from a genomic sequence that contains many introns.
search bar.
 Now click on the GenBank Feature Extractor on the right side down the SMS.

GenBank Trans Extractor
 GenBank Trans Extractor accepts a GenBank file as input and returns each of the protein
translations described in the file in FASTA format. GenBank Trans Extractor should be used when
you are more interested in the predicted protein translations of a DNA sequence than the DNA
sequence itself.
search bar.
 Now click on the GenBank Trans Extractor on the right side down the SMS.

One to Three
 One to Three converts single letter translations to three letter translations.
chrome search bar.
 Now click on the One to Three on the right side down the SMS.

Range Extractor DNA
 Range Extractor DNA accepts a DNA sequence along with a set of positions or ranges. The bases
corresponding to the positions or ranges are returned, either as a single new sequence, a set of
FASTA records, as uppercase text, or as lowercase text. Use Range Extractor DNA to obtain
subsequences using position information.
search bar.

 Now enter the base positions or ranges to be extracted. Use ".." to represent a range, and use a
can be included in the ranges. For example, to obtain the last three bases of a sequence, the range
'(end - 2)..end' can be used. To obtain the 30 bases on either side of the center base along with the
center base, the ranges '(center - 30)..(center - 1), center, (center + 1)..(center + 30)' can be used
and submit it.
Range Extractor Protein
 Range Extractor Protein accepts a protein sequence along with a set of positions or ranges. The
residues corresponding to the positions or ranges are returned, either as a single new sequence, a
set of FASTA records, as uppercase text, or as lowercase text. Use Range Extractor Protein to
obtain subsequences using position information.
search bar.

 Enter the residue positions or ranges to be extracted. Use "...” to represent a range, and use
a comma to separate entries. The words 'start', 'end', 'center', and 'length' can be used in
place of digits, to represent the beginning, end, middle, and length of the sequence.
Arithmetic expressions can be included in the ranges. For example, to obtain the last three
residues of a sequence, the range '(end - 2)..End' can be used. To obtain the 30 bases on
either side of the center residue along with the center residue, the ranges '(center -
30)..(center - 1), center, (center + 1)..(center + 30)' can be used and submit it.
13. Reverse Complement
Reverse Complement converts a DNA sequence into its reverse, complement, or reverse-
complement counterpart. The entire IUPAC DNA alphabet is supported, and the case of each
input sequence character is maintained. You may want to work with the reverse-complement of a
sequence if it contains an ORF on the reverse strand.
Steps:

 Open www.justbio.com.
 Log in and click SMS2.
 Click on the option “Reverse Complement”.
 Paste sequence 1, sequence 2 and sequence 3.
 Click “submit”.
 Reverse sequences are appeared.

14. Split Codons
Split Codons divides a coding sequence into three new sequences, each consisting of the bases
from one of the three codon positions. For example, the sequence "ATGATGATG" is converted
to "AAA", "TTT", and "GGG". Split Codons can be useful when you wish to analyse codon
positions separately in downstream analyses.

Steps:
 Click on the option “Split Codons”.
 Paste the sequence in the given box and click “submit”.
 Splitted codons with their positions are appeared.

15. Split FASTA
Split FASTA divides FASTA sequence records into smaller FASTA sequences of the size you
specify. An optional overlap value can be used to create sequences that overlap.
Steps:
 Click on the option “Split FASTA”.
 Paste the sequence in the given box.
 Smaller FASTA sequence fragments are appeared.

16. Three to One
Three to One converts three letter translations to single letter translations. Digits and blank
spaces are removed automatically. Non-standard triplets are ignored.
Steps:

 Click on the option “Three to One”.
 Paste sequence 1 and sequence 2 and click “submit”.
 The new cleaned translated sequence is appeared.

17. Window extractor DNA
Window Extractor DNA accepts a DNA sequence along with a window position and window
size. The bases corresponding to the window are returned, either as a new sequence, as uppercase
text, or as lowercase text. Use Window Extractor DNA to obtain subsequences using position
information.
Steps:
 Click on the option “Window extractor DNA”.
 Paste the DNA sequence in the given box.
 Subsequence of DNA with the position is appeared.

18. Window extractor Protein
Window Extractor Protein accepts a protein sequence along with a window position and window
size. The residues corresponding to the window are returned, either as a new sequence, as
uppercase text, or as lowercase text. Use Window Extractor Protein to obtain subsequences using
position information.
Steps:
 Click on the option “Window protein extractor”.
 Paste the protein sequence in the given box.
 The sub sequences with positions are appeared.

Sequence Analysis
Codon Plot
Codon Plot accepts a DNA sequence and generates a graphical plot consisting of a horizontal bar
for each codon. The length of the bar is proportional to the frequency of the codon in the codon
frequency table you enter. Use Codon Plot to find portions of DNA sequence that may be poorly
expressed or to view a graphic representation of a codon usage table (by using a DNA sequence
consisting of one of each codon type).
 Paste the raw or FASTA sequence into the text area below. Input limit is 50000 characters.

 Click on “Submit”.
 Codon plot result is obtained.
Codon Usage
Codon Usage accepts one or more DNA sequences and returns the number and
frequency of each codon type. Since the program also compares the frequencies of
codons that code for the same amino acid (synonymous codons), you can use it to
assess whether a sequence shows a preference for particular synonymous codons.

 Paste the raw sequence or one or more FASTA sequences into the text area. Input limit is
500000 characters.
 Click on ‘submit”.
 Codon usage result is obtained.

CpG Islands
CpG Islands reports potential CpG island regions using the method described by Gardiner-
Garden and Former (1987). The calculation is performed using a 200 bp window moving across
the sequence at 1 bp intervals. CpG islands are defined as sequence ranges where the Obs/Exp
value is greater than 0.6 and the GC content is greater than 50%. The expected number of CpG
dimers in a window is calculated as the number of 'C's in the window multiplied by the number
of 'G's in the window, divided by the window length. CpG islands are often found in the 5'
regions of vertebrate genes, therefore this program can be used to highlight potential genes in
genomic sequences.
 Paste the raw sequence or one or more FASTA sequences into the text area below. Input
limit is 100000 characters.
 Click on “submit”.
 CpG Islands result is obtained.

DNA Molecular Weight
DNA Molecular Weight accepts one or more DNA sequences and calculates molecular
weight. Sequences can be treated as double-stranded or single-stranded, and as linear or
circular. Single-stranded sequences are assumed to have a 5' phosphate. Use DNA Molecular
Weight when calculating molecule copy number.
 DNA Molecular Weight result is obtained.

DNA Pattern Find
DNA Pattern Find accepts one or more sequences along with a search pattern and returns
the number and positions of sites that match the pattern. Use DNA Pattern Find to locate
sequence regions that match a consensus sequence of interest.
 Paste a raw sequence or one or more FASTA sequences into the text area below.
White space and digits are removed before the pattern matching is performed. Input
 DNA Pattern find result is obtained.

DNA Stats
DNA Stats returns the number of occurrences of each residue in the sequence you enter.
Percentage totals are also given for each residue, and for certain groups of residues,
allowing you to quickly compare the results obtained for different sequences.

 DNA Stats result is obtained.
Fuzzy Search DNA
Fuzzy Search DNA accepts a DNA sequence along with a query sequence and returns sites that
are identical or similar to the query. You can use this program, for example, to find sequences
that can be easily mutated into a useful restriction site.

 Enter the sequence to search (in raw sequence or FASTA format) into the text area
below. Input limit is 2000 characters.
 DNA Stats result is obtained.

Fuzzy Search Protein
Fuzzy Search Protein accepts a protein sequence along with a query sequence and returns sites
that are identical or similar to the query.
 Enter any test protein sequence from library in the box given.
 Enter any query sequence in the second box.
 Click on Submit.
 Fuzzy Protein DNA results are obtained.

Ident and Sim
Ident and Sim accepts a group of aligned sequences (in FASTA or GDE format) and calculates
the identity and similarity of each sequence pair. Identity and similarity values are often used to
assess whether or not two sequences share a common ancestor or function.
 Paste any aligned sequence in FASTA format from library in the box given.
 Enter the groups of similar amino acids separated by commas to be used for the similarity
calculation in the second box.
 Click on Submit.Ident and Sim results are obtained.

Multi Rev Trans
Multi Rev Trans accepts a protein alignment and uses a codon usage table to generate a
degenerate DNA coding sequence. The program also returns a graph that can be used to find
regions of minimal degeneracy at the nucleotide level. Use Multi Rev Trans when designing
PCR primers to anneal to an un sequenced coding sequence from a related species.
 Paste the protein alignment in FASTA format into the text area given.
 Click on Submit.Multi Rev Trans results are obtained.

Mutate for Digest
Mutate for Digest accepts a DNA sequence as input and searches for regions that can easily be
mutated to create a restriction site of interest. The program also displays protein translations so
that you can see which reading frames are altered by the proposed mutations. Use Mutate for
Digest to find sequences that can be converted to a useful restriction site using PCR or site-
directed mutagenesis.
 Paste a raw sequence or one or more FASTA sequences into the text area given.
 Mutate for Digest results are obtained.

ORF Finder
ORF Finder searches for open reading frames (ORFs) in the DNA sequence you enter. The
program returns the range of each ORF, along with its protein translation. Use ORF Finder to
search newly sequenced DNA for potential protein encoding segments. ORF Finder supports the
entire IUPAC alphabet and several genetic codes.
 Paste the text into the text area given.

 ORF Finder results are obtained.
Pairwise Align Codons
Pairwise Align Codons accepts two coding sequences and determines the optimal global
alignment. The scoring matrix is used to calculate the alignment. Use Pairwise Align Codons to
look for conserved coding sequence regions. Only the bases A, C, G, T and U are used in the
alignment.
 Paste sequence one (in raw sequence or FASTA format) into the text area given.
 Paste sequence two (in raw sequence or FASTA format) into the text area given.
 Click on Submit. Pairwise Align Codons results are obtained.

Pairwise Align DNA
Pairwise Align DNA accepts two DNA sequences and determines the optimal global alignment.
Use Pairwise Align DNA to look for conserved sequence regions.
 Paste sequence one (in raw sequence or FASTA format) into the text area given.
 Paste sequence two (in raw sequence or FASTA format) into the text area given.
 Click on Submit. Pairwise Align DNA results are obtained.

Pairwise Align Protein
Pairwise Align Protein accepts two protein sequences and determines the optimal global
alignment. Use Pairwise Align Protein to look for conserved sequence regions.
Steps:
 Open Google chrome and go to the website of Justbio.com.
 A page opens. Click on the website of Cellbio.com given on the left side of page below
login option.
 A new page opens. Click on the SMS2 tool that is highlighted green.
 Several tools appear on the left side of the page. Click on Pairwise Align Protein tool
under the heading of Sequence Analysis.
 In the page opened paste in protein sequences in the given boxes and click on the
Submit button.
 After submission the following alignment score appears.

PCR Primer Stats
PCR Primer Stats accepts a list of PCR primer sequences and returns a report describing the
properties of each primer, including melting temperature, percent GC content, and PCR
suitability. Use PCR Primer Stats to evaluate potential PCR primers.
Steps:
 Go back to the SMS2 tools. Click on the option of PCR Primer Stats.
 A new page opens. Copy a protein sequence and paste it in the given box.
 Click on the Submit button.
 The following results appear after submission.

PCR Products
PCR Products accepts one or more DNA sequence templates and two primer sequences.
The program searches for perfectly matching primer annealing sites that can generate a
PCR product. Any resulting products are sorted by size, and they are given a title
specifying their length, their position in the original sequence, and the primers that
produced them. You can use linear or circular molecules as the template. Use PCR
Products to determine the product sizes you can expect to see when you perform PCR in
the lab.
Steps:
 Go to the SMS2 tools and click on the PCR Product option.
 A new page opens. Copy a DNA sequence and paste it in the given box.

 The following PCR results are obtained.
Protein GRAVY
Protein GRAVY returns the GRAVY (grand average of hydropathy) value for the protein
sequences you enter. The GRAVY value is calculated by adding the hydropathy value for
each residue and dividing by the length of the sequence (Kyte and Doolittle; 1982).
Steps:
 Go to the SMS2 tool and click on the option of Protein GRAVY.
 A new page opens. Copy a protein sequence and paste it in the given box.

 The following results are obtained.
Protein Isoelectric Point
Protein Isoelectric Point calculates the theoretical pI (isoelectric point) for the protein
sequence you enter. Use Protein Isoelectric Point when you want to know approximately
where on a 2-D gel a particular protein will be found.
Steps:
 Go to the SMS2 tools and click on the option of Protein Isoelectric Point.
 A new page opens. Copy two protein sequences from your library and paste them in the
given box.

 The following results are obtained.
Protein Molecular Weight
Protein Molecular Weight accepts one or more protein sequences and calculates
molecular weight. You can append copies of commonly used epitopes and fusion proteins
using the supplied list. Use Protein Molecular Weight when you wish to predict the
location of a protein of interest on a gel in relation to a set of protein standards.
Steps:
 Go to the SMS2 tools and click on the option of Protein Molecular Weight.
 A new page opens. Copy two protein sequences from your library and paste them in the
given box.

 The following results are obtained after submission.
Protein pattern find:
Protein Pattern Find accepts one or more sequences along with a search pattern and returns the
number and positions of sites that match the pattern. Use Protein Pattern Find to locate sequence
regions that match a consensus sequence of interest.

Steps:
 Go to www.justbio.com
 Go to www.cellbio.com and click on SMS2
 Click on “Protein Pattern Find”
 Paste protein sequence here.
 Click on “Submit”
 Results will be visible.

Protein stats:
Protein Stats returns the number of occurrences of each residue in the sequence you enter.
Percentage totals are also given for each residue, and for certain groups of residues, allowing you
to quickly compare the results obtained for different sequences.

Steps:
 Click on “Protein Stats”
 Results will be shown after processing.

Restriction digest:
Restriction Digest cleaves a DNA sequence in a virtual restriction digest, with one, two, or three
restriction enzymes. The resulting fragments are sorted by size, and they are given a title
specifying their length, their position in the original sequence, and the enzyme sites that
produced them. You can digest linear or circular molecules, and even a mixture of molecules (by
entering more than one sequence in FASTA format).

Steps:
 Click on “Restriction Digest”
 Paste DNA sequence here.
 Results will be shown after processing.

Restriction Summary:
Restriction Summary accepts a DNA sequence and returns the number and positions of
commonly used restriction endonuclease cut sites. Use this program if you wish to quickly
determine whether or not an enzyme cuts a particular segment of DNA.

Steps:
 Click on “Restriction Summary”
 Restriction endonucleases cut sites will be visible here.

Reverse translate:
Reverse Translate accepts a protein sequence as input and uses a codon usage table to generate a
DNA sequence representing the most likely non-degenerate coding sequence. A consensus
sequence derived from all the possible codons for each amino acid is also returned. Use Reverse
Translate when designing PCR primers to anneal to an unsequenced coding sequence from a
related species.

Steps:
 Click on “Reverse Translate”
 Protein thus translated into respective DNA sequence.

Translate:
Translate accepts a DNA sequence and converts it into a protein in the reading frame you
specify. Translate supports the entire IUPAC alphabet and several genetic codes.

Steps:
 Click on “Translate”
 DNA sequence will be translated into protein.

Sequence Figures
1. Colour align conservation
Colour Align Conservation accepts a group of aligned sequences (in FASTA or GDE format)
and colours the alignment. The program examines each residue and compares it to the other
residues in the same column. Residues that are identical among the sequences are given a black
background, and those that are similar among the sequences are given a grey background. The
remaining residues receive a white background. You can specify the percentage of residues that
must be identical and similar for the colouring to be applied. Use Colour Align Conservation to
enhance the output of sequence alignment programs.
Steps:
 Opened the colour align conservation.
 Entered the sequence.

 Click on submit.
 The entered sequences were aligned in colours.
2. Colour align properties
Colour Align Properties accepts a group of aligned sequences (in FASTA or GDE format)
and colours the alignment. The program examines each residue and compares it to the other
residues in the same column. Residues that are identical or similar among the sequences are
given a coloured background. The colour is chosen according to the biochemical properties
of the residue. You can specify the percentage of residues that must be identical and similar
for the colouring to be applied. Use Colour Align Properties to highlight protein regions with
conserved biochemical properties.

Steps:
 Opened Group DNA.
 Clicked on submit.
 Results appeared.

3. Group protein
Group Protein adjusts the spacing of protein sequences and adds numbering. You can specify the
group size (the number of residues per group), as well as the number of residues per line. The
output of this program can serve as a convenient reference, since the numbering and spacing
allows you to quickly locate specific residues.
Steps:
 Opened Group protein.

4. Primer map
Primer Map accepts a DNA sequence and returns a textual map showing the annealing
positions of PCR primers. Restriction endonuclease cut sites, and the protein translations of
the DNA sequence can also be shown. Use this program to produce a useful reference figure,
particularly when you have designed a large number of primers for a particular template.
Primer Map supports the entire IUPAC alphabet and several genetic codes.
Steps:
 Entered the one or more raw sequences in FASTA format.
 Entered the primers required.

5. Restriction map
Restriction Map accepts a DNA sequence and returns a textual map showing the positions of
restriction endonuclease cut sites. The translation of the DNA sequence is also given, in the
reading frame you specify. Use the output of this program as a reference when planning
cloning strategies. Restriction Map supports the entire IUPAC alphabet and several genetic
codes.

Steps:
 Opened the restriction map.
 Entered the one or more sequences in FASTA format.

6. Translation Map
Translation Map accepts a DNA sequence and returns a textual map displaying protein
translations. The reading frame of the translation can be specified (1, 2, 3, or all three), or
you can choose to treat uppercase text as the reading frame. Translation Map supports the
entire IUPAC alphabet and several genetic codes.
Steps:
 Opened the Translation map.
 Entered the DNA sequence.

RANDOM SEQUENCING
Mutate DNA
 Mutate DNA introduces base changes into a DNA sequence. You can select the number
of mutations to introduce, and whether or not to preserve the first and last three bases in
the sequence, to reflect selection acting to maintain start and stop codons. The position of
each mutation is chosen randomly, and multiple mutations can occur at a single site.
Mutated sequences can be used to evaluate the significance of sequence analysis results.
 Click on mutate DNA.
 Following page will appear.
 Paste DNA sequences from your library.
 Click on submit. Following result will appear.

Mutate proteins
 Mutate Protein introduces residue changes into a protein sequence. You can select the
number of mutations to introduce, and whether or not to preserve the first residue in the
sequence, to reflect selection acting to maintain a start codon. The position of each
mutation is chosen randomly, and multiple mutations can occur at a single site. Mutated
sequences can be used to evaluate the significance of sequence analysis results.
 Click on Mutate proteins

 Click on submit and result will be given as

Random Coding DNA
 Random Coding DNA generates a random open reading frame beginning with a start
codon and ending with a stop codon. You can choose the genetic code to use and the
length of the sequence to generate. Random sequences can be used to evaluate the
significance of sequence analysis results.
 Click on Random Coding DNA
 Click on submit and result will appear as:

Random DNA Sequence
 Random DNA Sequence generates a random sequence of the length you specify. Random
 Click on Random DNA Sequence.
 And a page will appear as:

 Click on submit on above picture.
 And result will appear as given in following picture.

Random DNA Regions
 Random DNA Regions replaces regions of a DNA sequence with random bases. Random
 Click on Random DNA Regions.
 Click on submit in above picture.
 Result will appear as:

Random Protein Sequence
 Random Protein Sequence generates a random sequence of the length you specify.
Random sequences can be used to evaluate the significance of sequence analysis results.
 Click on Random Protein Sequence.
 A page will appear as:

 Result will come as:

Random Protein Regions
 Random Protein Regions replaces regions of a protein sequence with random residues.
Random sequences can be used to evaluate the significance of sequence analysis results.
 Click on Random Protein Regions.
 A page will be given as:
 Result will appear as given in following picture:

Sample DNA
 Sample DNA randomly selects bases from the guide sequence until a sequence of the
length you specify is constructed. Each selected base is replaced so that it can be selected
again.
 Click on sample DNA.
 Following page will appear:

 So the sample DNA result will be:

Sample Protein
 Sample Protein randomly selects bases from the guide sequence until a sequence of the
length you specify is constructed. Each selected residue is replaced so that it can be
selected again.
 Click on Sample Protein. Following page will appear.
 And click on submit.

Shuffle DNA
 Shuffle DNA randomly shuffles a DNA sequence. Shuffled sequences can be used to
evaluate the significance of sequence analysis results, particularly when sequence
composition is an important consideration.
 Click on Shuffle DNA.

 Following result will come as:

Shuffle Protein
 Shuffle Protein randomly shuffles a protein sequence. Shuffled sequences can be used to
evaluate the significance of sequence analysis results, particularly when sequence
composition is an important consideration.
 Click on Shuffle Protein.
 Page will appear as:
 Result will come as:

Miscellaneous
1. IUPAC codes
It tells IUPAC codes of DNA and Proteins.
Steps:
 Clicked on IUPAC codes.
 Then table of IUPAC codes of DNA and protein appeared separately as shown below.
IUPAC codes of DNA

IUPAC codes of protein
2. Genetic codes:
The instructions in a gene that tell the cell how to make a specific protein. A, T, G, and C are the 'letters'
of the DNA code and represent the chemicals adenine, thymine, guanine, and cytosine, respectively.
These make up the nucleotide bases of DNA. Each gene's code combines these four chemicals in various
ways to spell out three-letter 'words' that specify which amino acid is needed at every step in making a
protein.
Steps:
 Clicked on genetic codes.
 Then genetic codes appeared, as shown below.

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Phylogeny.fr
Robust Phylogenetic Analysis for the Non-Specialist
Phylogeny.fr is a free, simple to use web service dedicated to reconstructing and analysing
phylogenetic relationships between molecular sequences.Phylogeny.fr runs and connects
various bioinformatics programs to reconstruct a robust phylogenetic tree from a set of
sequences.
Phylogeny.fr has been designed to provide a high performance platform that transparently
chains programs relevant to phylogenetic analysis in a comprehensive, and flexible pipeline.
Although phylogenetic aficionados will be able to find most of their favourite tools and run
sophisticated analysis, the primary philosophy of Phylogeny.fr is to assist biologists with no
experience in phylogeny in analysing their data in a robust way.
 First of all open the Google chrome and type http://www.phylogeny.fr/index.cgi on
search bar.
 Then click on www.phylogeny.fr/index.cgi and then the Phylogeny.fr page will open.
Phylogeny Analysis
The Phylogeny.fr platform offers a phylogeny pipeline which can be executed through three
main modes:
One Click Mode

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The "One Click mode" targets users that do not wish to deal with program and parameter
selection. By default, the pipeline is already set up to run and connect programs recognized
for their accuracy and speed (MUSCLE for multiple alignment and PhyML for phylogeny) to
reconstruct a robust phylogenetic tree from a set of sequences.
 Click on the Phylogeny Analysis on the tool bar of the Phylogeny.fr.
 A list of further tools will appear on that phylogeny analysis.
 Select these one by on as first click on the “One Click”
 The one click mode will be appeared on the screen as given in the following figure.

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 Now paste your sequence in the given bar.
 Then click on Submit.
 Alignment results of the one click mode will going to proceed and it takes a few
seconds to complete the alignment.

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 After aligning, the phylogeny results will appear on the screen and also takes a few
seconds to proceed.
 In the end, rendering results of one click of phylogeny analysis will be obtained.
 Tree like structure of the inserted sequences will be formed.

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Advanced Mode
In the "Advanced mode", the Phylogeny.fr server proposes the succession of the same
programs but users can choose the steps to perform (multiple sequence alignment,
phylogenetic reconstruction, tree drawing) and the options of each program.

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 Click on the “Advanced”.
 The Advanced mode will be appeared on the screen as given in the following figure.
 The workflow settings will appear. Now click on create workflow.
 Paste the sequence which was selected through the library in that input data bar.
 Then click on the submit option.

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 Alignment results of the advanced mode will going to proceed and it takes a few
 After aligning, the phylogeny results will appear on the screen and also takes a few
seconds to proceed.

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 In the end, rendering results of advanced mode of phylogeny analysis will be
obtained.

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A la carte mode
The "A la carte mode" offers the possibility of running and testing more alignment and
phylogeny programs, such as MUSCLE, ClustalW, T-Coffee, PhyML, BioNJ, TNT,...
Alternatively, users have the possibility to run the different programs separately.
 Click on the “A la carte”.
 The A la carte mode will be appeared on the screen as given in the following figure.

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 The workflow settings will appear. Now click on create workflow.
 Alignment results of the A la carte mode will going to proceed and it takes a few

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 In the end, rendering results of A la carte mode of phylogeny analysis will be
obtained.

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Blast Explorer
A fast BLAST search on Giga blaster allows to quickly explore your sequence neighbours.
Paste your single sequence, run blast and explore its homologous sequences. The system
facilitates the selection of homologous sequences, based on a 'quick-and-dirty' phylogenetic
representation using BLAST results and an estimator of the final multiple alignment length.
 Click on the Blast Explore on the tool bar of the Phylogeny.fr.
 Blast (doc) will appear now select the suitable program on it.

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 Alignment results of the A la carte mode will going to proceed and it takes a few
 The blast occurs that results in exploring the inserted sequence neighbours.

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Online Programs
In online programs the first option is blast which is similar to that of Blast Explorer. The next
block is of Multiple Alignment, in which further options are present as below:
Muscle

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MUSCLE is a program for creating multiple alignments of amino acid or nucleotide sequences.
A range of options is provided that give you the choice of optimizing accuracy, speed, or
some compromise between the two. Default parameters are those that give the best average
accuracy in our tests. Using versions current at the time of writing, tests show that MUSCLE
can achieve both better average accuracy and better speed than CLUSTALW or T-Coffee,
depending on the chosen options.
 Click on the online programs on the tool bar of the Phylogeny.fr.
 A list of further tools will appear on that online programs.
 Select these one by on and first click on the “MUSCLE”
 The one click mode will be appeared on the screen as given in the following figure.
 MUSCLE (doc) will appear.

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 Alignment results of the Muscle will going to proceed and it takes a few seconds to
complete the alignment.

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 After that the similar residues will appear in the colour so the muscle alignment result
will be obtained.
T-Coffee

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A collection of tools for Computing, Evaluating and Manipulating Multiple Alignments of
DNA, RNA, Protein Sequences and Structures.
 Click on the T-Coffee on the multiple alignment.
 The T-Coffee mode will be appeared on the screen as given in the following figure.
 T-Coffee (doc) will appear.

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 Alignment results of the T-Coffee will going to proceed and it takes a few seconds to

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 Alignment result of the T-Coffee will be appeared on the screen.

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ClustalW
All variants of Clustal align sequences by three main steps:
1. Do a pairwise alignment
2. Create a guide tree (or use a user-defined tree)
3. Use the guide tree to carry out a multiple alignment
These are done automatically when you select "Do Complete Alignment". Other options are
"Do Alignment from guide tree and phylogeny" and "Produce guide tree only".
 Click on the ClustalW on the multiple alignment.

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 The ClustalW mode will be appeared on the screen as given in the following figure.
 ClustalW (doc) will appear.
 Alignment results of the ClustalW will going to proceed and it takes a few seconds to

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 Alignment result of the ClustalW will be appeared on the screen.
ProbCons
PROBCONS is a novel tool for generating multiple alignments of protein sequences. Using a
combination of probabilistic modelling and consistency-based alignment techniques,
PROBCONS has achieved the highest accuracies of all alignment methods to date.

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 Click on the PROBCONS on the multiple alignment.
 The PROBCONS mode will be appeared on the screen as given in the following
figure.
 PROBCONS (doc) will appear.

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 Alignment results of the ProbCons will going to proceed and it takes a few seconds to
 Alignment result of the ProbCons will be appeared on the screen.

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PhyML
PhyML is a phylogeny software based on the maximum-likelihood principle. Early PhyML
versions used a fast algorithm to perform Nearest Neighbour Interchanges (NNIs), in order to
improve a reasonable starting tree topology. Since the original publication, PhyML has been
widely used (>1,250 citations in ISI Web of Science), due to its simplicity and a fair
accuracy/speed compromise.
 Click on the PhyML on the Phylogeny.

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 The PhyML mode will be appeared on the screen as given in the following figure.
 PhyML (doc) will appear.
 Phylogeny of PhyLM will be occurred which will take a few seconds in processing.

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 The result of PhyML will be as follows:
BioNJ
BIONJ is well suited for distances estimated from DNA or protein sequences. BIONJ has
better topological accuracy than NJ in all evolutionary conditions; its superiority becomes

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important when the substitution rates are high and varying among lineages. However, BIONJ
and NJ trees are often close or identical, notably when the number of taxa is low. BIONJ
retains the speed of NJ and can be applied to very large data sets (> 1000 taxa). BIONJ can be
run on several matrices to build bootstrap trees. As usual, distances should be corrected, e.g.
using DNADIST or PROTDIST from PHYLIP.
 Paste the sequence in the given block and click on submit.
 Phylogeny of BioNJ will be occurred which will take a few seconds in processing.

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 The result of BioNJ will be as follows:
TNT
It is a program for phylogenetic analysis under parsimony, as well as extensive tree handling
and diagnosis capabilities.

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 Phylogeny of TNT will be occurred which will take a few seconds in processing.
 The result of TNT will be as follows:

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TreeDyn
 The result of TreeDyn will be as follows:

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Drawgram
 The result of Drawgram will be as follows:

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Drawtree
 The result of Drawtree will be as follows:

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Gblocks
 The result of Gblocks will be as follows:

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