Bioinformatics Tutlets

1. Edit the sequence header as shown to include your own database id (eg.STAT_00044 from my database) in the fasta file

>gi|34392270|emb|CAE46396.1| STAT_00044 Dd-STATb protein [Dictyostelium discoideum]

2. formatdb

formatdb -i input.fasta -p T -o T

3. In the /usr/local/wwwblast-2.2.17 folder of BioSLAX, backup blast.html and blast.cgi

4. Change the content of blast.cgi as shown (pipe the output of blast.REAL and do sed to get the hyperlink of your own database Id included in the blast result)

#!/bin/csh -f

#
# $Id: blast.cgi,v 1.1 2002/08/06 19:03:51 dondosha Exp $
#

echo "Content-type: text/html"
echo ""

#setenv DEBUG_COMMAND_LINE TRUE
setenv BLASTDB db

# I used my own database STATdb_Id as an example below.

5. Output example

39) How To Create a Password Protected Website

2010-03-11T20:40:00.001+08:00

howtocreateapasswordprotectedwebsite

38) Running Blast+ client on Mac OSX

2010-03-08T03:58:00.003+08:00

runningblast+clientonmac

37) LAPIS: Converting blast hit sequences into fasta format

2009-12-14T19:52:00.020+08:00

LAPIS is an Open Source software written in JAVA. One of its uses is to make data presentation simpler for analysis by extracting suitable information from a text file.

For example, it can be used to extract only the subject and the identifier (NCBI GI number) information from a BLAST result. Below is an illustration of how this can be done using LAPIS:

Before

>gi|126385999|gb|CP000521.1| Acinetobacter baumannii ATCC 17978, complete genome
Length = 3976747

Score = 570 bits (1470), Expect = e-163
Identities = 284/284 (100%), Positives = 284/284 (100%)
Frame = -2

Query: 1 LNFKFNFISLMNIKALLLITSAIFISACSPYIVTANPNHSASKSDEKAEKIKNLFNEAHT 60
LNFKFNFISLMNIKALLLITSAIFISACSPYIVTANPNHSASKSDEKAEKIKNLFNEAHT
Sbjct: 1766322 LNFKFNFISLMNIKALLLITSAIFISACSPYIVTANPNHSASKSDEKAEKIKNLFNEAHT 1766143

After

>gi|126385999
LNFKFNFISLMNIKALLLITSAIFISACSPYIVTANPNHSASKSDEKAEKIKNLFNEAHT

Methodology:
1. Select the line containing the fasta description together with the line containing the subject sequences: “line containing > or line containing sbjct” ->Tools ->Extract
2. To get rid of the numbers in the line containing sbjct: “digits in line containing sbjct” ->Tools -> Omit
3. To get rid of sbjct: “sbjct:” -> Extract -> Omit
4. To get rid of dashes: type “-" -> Extract -> Omit
5. To get rid of the extra spaces in the lines containing sequences: “spaces not in line containing >” -> Tools -> Omit
6. In case you want to clean up the description line to only have the GI
From second | in line containing > to start of linebreak

Screen shots: The following screen shots shows the input and the output at each step

For extracting specific information, the user needs to find a pattern and type it in the pattern box as shown below:

The pattern above is used to extract the two necessary lines for the further analysis.

Next the user should remove the positions (digits) in the subject line.

The screen shot below shows the highlighted digits to be omitted.

The screen shot below shows the information after omitting the numbers from the subject line.

Next the word “subj:” should be omitted also.

The screen shot below shows the pattern to remove any extra spaces in the subject line. In case of any gaps (-), they should also be removed.

The screen shot below shows the pattern to remove extra information from the header line.

The screen shot below shows the required output.

36) OpenBabel GUI - file format converter

2009-09-27T19:33:00.012+08:00

It is a chemical toolbox designed to speak the many languages of chemical data.

OpenBabel GUI for windows can be downloaded from the following website,

http://www.brothersoft.com/open-babel-download-208843.html,

OpenBabel is also available for LINUX and MACINTOSH,

Basic operation:

OpenBabel converts chemical objects (currently molecules or reactions) from one file format to another. This interface is an alternative to a command line and has the same capabilities.

- Select the type of the type of the input file from the dropdown list.

- Click the "..." button and select the file. Its contents are displayed.

- Choose the output format and file in a similar way. You can merely display the output without saving it by not selecting an output file or by checking "Output below only..".

- Click the "Convert" button.

The message window below the button gives the number of molecules converted, and the contents of the output file are displayed.

By default, all the molecules in an input file are converted if the output format allows multiple molecules.

NOTE: This would be useful for the people who are handling different docking softwares, because the file formats for each docking software will differ, based on the type of algorithm used and the development of the software.

35) Autodock 4.0 (Installation and working)

2009-09-26T23:28:00.010+08:00

Autodock 4.0 can be found from the following website,

http://mgltools.scripps.edu/downloads

download the software according to your computer appropriate operating system,

Installation:

I.windows installation:

1.Double click on to the MGLTools setup file.

2.Click "Next"

3.Accept the license agreement

4. Select your favorite destination folder to install the files and click 'Next"

5. wait for a while for the files to get installed

6. click on to the "Non commercial-Install all these packages"

7. click on to each package license

8. click "continue" button below

9. If you wish to launch shortcuts on your desktop , you can just click "finish"

10. And there your are ---- you can start working.

II. Linux installation:

NOTE: Autodock need to install from /root/, it can be installed from the /home/ directory itself.

1. Download MGLTools-1.5.2-Linux-x86-Install grant execute permission and run the installer.
To grant execute permission, type the following command at a command prompt: chmod +x MGLTools-1.5.2-Linux-x86-Install .
You can also use file manager to grant execute permission (right-click on the downloaded file and select Properties).

2) In the install wizard, click Next, select the destination and click Next again.
The default destination is /usr/local/MGLTools-1.5.2 for the root user and $HOME/MGLTools-1.5.2 for all others.
Please wait a few moments until the "License Agreements for add-on packages" window appears.

3) Complete the "License Agreements for add-on packages" form.
See Filling add-on packages license form for details.

4) Finally, click Finish to complete installation,

The README file contains detailed information on how to setup your environmental variables for command line usage.

Meanwhile download the following as well,

http://autodock.scripps.edu/downloads

click on the above link,

you will pop up with an registration form, kindly fill with necessary details and download the files which will be compatible to your operating system that your using.

This will contain 2 executable files,

1. autogrid

2.autodock.

NOTE: Make sure you install everything in the same destination folder including the 2 executable files

How to work on Autodock 4.0?

Here we go..!!

Follow the below steps,

*double click on to this icon

Your workspace will look like this,

Kindly use the pdb and ligand files which comes along with the installation of Autodock 4.0, will be present in (MGLTools/MGL ToolsPckgs/Pmv/doc). Use hsg1.pdb (protein) and ind.pdb (ligand)

Protein Preparation:

File -  Read Molecule - Open the file.pdb.

Select - Select from string - Type HOH* (to remove water molecules)

Edit -  Add  Polar only

File - write PDB  save ur file in pdbfile.pdb extension just without changing the path.  just click and remove HETATM  Click sort nodes before saving .

Ligand Preparation:

Ligand -   input  open ur ligand.pdb - just note down the summary of the ligand.

Torsion tree -  choose root

again go to torsion tree - detect root

again go to torsion tree - show root expansion

again go to torsion tree - choose torsions  just click ok

again go to torison tree - set number of torsions  give the number of torisons that you get in the summary of the (TORSDOF) when u open the ligand.

Output  Save the ligand file in the same directory in ligandfile.pdbqt

Grid preparation:

Select  select from string  just type your active site residues and then click ok

Flexible residues - input macromolecule - choose macromolecule - click on your pdb from the panel displayed.

Flexible residues - choose torsions from the currently selected residues - active site residues alone will be highlighted.

Flexible residues - redisplay the macromolecule

Flexible residues - save the files in both    flexible as (pdbfile_flex.pdbqt)   rigid as (pdbfile_rigid.pdbqt) .

Grid - macromolecule  - open- just open ur pdb_rigid.pdbqt file, ignore the warnings, if you have any metal ions in your  proteinn ,  just open ur grid file and u can find (0.00) for ur metal ions, just find the valency for them from net  and type maunally ther and again save it.you use this now. (if you dont hav metal ions), just ignore the warnings.

set map types - choose ligand - click on ur ligand from the panel displayed  output - save in .gpf  edit gpf - dontt do anything just click ok for the time being

Run -  run autogrid  just browse and locate ur autogrid.exe file , just browse and locate file .gpf (grid parameter file) and note that everything should be in the same directory, were you saved your pdb in protien preparation, dont change the path for your wish, just save were it is saving  just put your autodock.exe and autogrid.exe in the same directory were ur files has been saved

Docking:

Docking -  Macromolecule - set rigid file name - just again open ur pdb_rigid.pdbqt file  Ligand - choose ur ligand  search parameters- just delete one zero from the 2500000 (just make it to 4 zeros), otherwise it wil take very long time.

Docking parameters- default  other option-autodock 4 parameters- default  output - save in ligandfile.dpf

Edit DPF - just leave and click ok

Run - run autock  just browse and locate ur autodock.exe file  just browse and locate file .dpf (dock parameter file) and then click ok.

Analysing the results:

open the widget "Analyse" and click open, locate the ligand.dlg file,

Next click on to Macromolecule found below Dockings, just click open, it will open by default, if you cannot able to view your protein properly, simply press "N" button from the keyboard.

To view your results click on to conformations -play,


It will open a widget shown in the picture.

click on to the ampersand (&) symbol.


Analyse your results..!!

NOTE: kindly save all your files in the same folder where you have been installed your MGLTools and executable files, if you want to run the programme from your favourite folder of your choice, make sure that you place just the executable files alone in the place were you have saved your all  files that you have been used during the course of working on Autodock, because without these executable files the programme wont run properly.

For more details regarding the algorithm,f orcefields and interpretation of the data kindly go through the Autodock manual which is available at below mentioned web site,

http://autodock.scripps.edu/faqs-help/tutorial/using-autodock-4-with-autodocktools

34) How to set up web blast using BioSlax

2009-09-26T23:16:00.001+08:00

At this stage you should have the bioslax installed, if not please refer to

A guide to using BioSlax and/or Installing BioSlax as a full linux system to your hard drive

1. First, get your sequence dataset ready in FASTA format

2. Create a directory (for example: /root/Desktop/db) and save the FASTA file of interest to this newly created directory

3. Now convert the FASTA formatted sequences into a blastable format, to do this, use the program “formatdb” which available for download from NCBI at http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=Download

*For BioSlax user, you do not need to download formatdb for BioSlax because it comes preinstalled

4. At console terminal, use this command to invoke Formatdb:
formatdb –i -p
Examples:
#formatdb –i ecoli.nt –p F
#formatdb -i swissprot -p T -o T

–p => type of sequence file;
T(rue) => the fasta file contains protein sequences
F(alse) => the fasta file contains nucleotide sequences
-o T => create an index file so that it is faster to run BLAST (optional)

If the command is successful, a bunch of new files are created.

5. Now, go to K-menu => BioSLAX => Desktop Apps => BLAST DB setup

6. Fill up or browse the path of the directory containing the formatdb formatted sequence files (the directory you created at part 1) => click on “Set BLAST DB Now!” for Web Blast to do the necessary setup configurations

* Please note that the example “/root/Desktop/db” directory is fine if it is on a hard disk or a “configsave” has been done. If it's on a machine booted by LiveCD & no “configsave” is done, the files will be lost when the system is shutdown. Therefore, we recommend that user to create the database outside the virtual environment, i.e in a USB hdd or thumb drive.

7. We are done, below are the results you will see:

BLAST result page

The location of your database after setting up BLAST Db: /usr/local/wwwblast-2.2.17/db

33) How to set-up the bioslax laptop servers

2009-09-26T23:16:00.000+08:00

At this step you should have the bioslax installed in your laptop, if not please refer to
A guide to using BioSlax and/or Installing BioSlax as a full linux system to your hard drive

1. First of all, you need to change the password:

Run the Konsole terminal
bioslax#: passwd
follow the instructions on the screen (please don’t set simple password)

2. To change host name: (skip this step if it has already been done)

Run the Konsole terminal
root@slax: pico /etc/HOSTNAME
Change “bioslax.maxonline.com.sg” to your host+domain name (e.g.group01.bic.nus.edu.sg).
Reboot

3. If the previous step 2 did not work, do the following, otherwise skip to step 4:

Run the Konsole terminal
bioslax#: cd /etc/rc.d
bioslax#: pico rc.M
You will be in the file rc.M. Type ctrl-W to search for "HOSTNAME". Type ctrl-W again, press enter and it will search the next word containing "HOSTNAME". You should be looking for the line "Set hostname by using DHCP response".
Comment the whole paragraph from "set hostname by using DHCP response" to "done" by typing # at the start of each line.
Save the changes by typing ctrl-X.
Run the Konsole terminal again.
bioslax#: netconfig
There will be a blue window opened where you can enter the hostname, e.g."group01".
Enter the domain name, e.g."bic.nus.edu.sg".
Select "DHCP" as the network connection.
At the "Set DHCP Hostname" window, leave it blank and press OK.
Confirm the changes.
Reboot

4. Set password for MYSQL

Log in to Webmin (e.g. type http://group01.bic.nus.edu.sg:10000 for remote access) in Mozilla Firefox.
Go to Servers
Select MySQL Database Server
Go to User Permissions
Search for the row containing user “Anonymous”, host “localhost” and click on the "Anonymous" link to change the settings for that row:
Under “Permissions” Click “Select Table Data” (it will become highlighted) Click “Save”
Search for the row containing user “Anonymous”, host “bioslax” and click on the "Anonymous" link to change the settings for that row:
Under “Hosts” click the radio button next to the text-box and in the textbox type in “groupXX”
Under “Permissions” Click “Select Table Data” (it will become highlighted)
Click “Save”
Search for the row containing user “root”, host “localhost” and click on the "Anonymous" link to change the settings for that row:
Under “Password” click the radio button for “Set to” and fill in the root password in the text box
Click “Save”
Search for the row containing user “root”, host “bioslax” and click on the "Anonymous" link to change the settings for that row:
Under “Password” click the radio button for “Set to” and fill in the root password in the text box
Under “Hosts” click the radio button next to the text-box and in the textbox type in “groupXX”
Click “Save”

5. Three different ways to access the server remotely

WinSCP (Freeware; http://winscp.net/).
1. Hostname: group02.bic.nus.edu.sg
2. Port number: 22
3. Username: root (or any other username that you may have created on the server)
4. Password: (type your password)
5. Protocol: SFTP (allow SCP fallback)
6. Click “Login”. If prompted for security alert, click “Yes”.
Putty (Freeware; http://www.putty.nl/).
1. Hostname: group02.bic.nus.edu.sg
2. Port number: 22
3. Connection type: SSH
4. Click “Open”. If prompted for security alert, click “Yes”.
5. Login as: root (or any other user id)
6. Password: (type your password)
Web browser (URL:http://group02.bic.nus.edu.sg)
1. To access files from the web, you should save your web pages in the “/var/www/htdocs” folder
2. To access pre-installed programs in Bioslax, such as blast and clustalw:

32) Duplicates Finder - a java standalone application to find sequence duplicates in your dataset

2009-09-02T16:04:00.016+08:00

Protein (Nucleotide) Sequences downloaded from NCBI Taxonomy Browser or other similar databases for an organism, in our case viruses, contain sequences which are duplicates of each other. Sometimes, these sequence duplicates turn out to be duplicate GenPept records, as found during our analyses, which need to be removed from the dataset to remove bias.

One could use the ‘Remove duplicates’ function in Excel to do this; it returns the list of unique sequences. However, Excel does not do a good job when the dataset is large (large number or long sequences).

Here, we provide a simple desktop application to extract the sets of duplicates from a dataset and also provide the list of unique sequences from the input dataset.

How to use the program:

Download the zip file DuplicateFinder.zip here. Extract contents to a desired location.

Click on the DuplicatesFinder.jar, the program starts and you are good to go. Since this is a java program, it is OS independent.
(NOTE: You need to have java runtime environment (JRE) installed in your system to run the program. To check if your system has java installed, refer to the end of this post.)

Input Files accepted by the program:

Comma separated values (*.csv)
- The data should contain two colums – Identifier number, Sequence.
  Identifier is a unique number to identify the sequence.
  Sequence is the set of sequences to be analyzed.

FASTA (*.fasta) or Tab delimited (*.txt)
- The sequences should be in fasta format as shown below
  >716349
  uquiertyqyjej
  quiejhieq
  >837468
  oiweirjtyiwj

Output Files created by the program:
The output files are created in the same directory and folder as the input file.
Two output files are created:

Duplicates sequences File: A file containing the sets of duplicates (
Duplicates.csv). The columns in the file are:
- Duplicate Set – the index of groups of duplicates;
- Count – the index of a duplicate sequence in the set of duplicates it belongs to. So each series of numbers from 1 to n represents a set of sequence duplicates with a total of n duplicate sequences in that set;
- Sequence – the duplicate sequences in ascending order (for easy visual checking).
- Identifier – the number from the identifier column in the .csv file or the identifier number after '>' in the .fas and .txt files.
- Remarks – remarks if any.

Unique sequences file: A file containing the unique sequences from the dataset in fasta format (Unique.fas). The file includes one representative from each set of duplicates.

NOTE: In both output files, the sequences are arranged in ascending order of the sequence.

A Working Example

Input file with 3 unique sequences and 2 groups of duplicates
(Group 1 – 1A and 1B; Group 2 – 2A, 2B and 2C)

Start the Duplicate Finder program. Click ‘Open’. A file chooser with file filtering set to show only *.csv, *.fas or *.txt files pops up. Select your input file (here, Sample.fas). Click ‘Open’.

The path of your input file is set. Click ‘Generate results’. Wait for the confirmation window saying “Done” to indicate that your results have been generated. Click ‘OK’.

Go to the folder containing your input file. You will find two new files created with names Duplicates.csv and Unique.fas. In our case, our input file was Sample. So the results are SampleDuplicates.csv and SampleUnique.fas.

Output file 1: SampleDuplicates.csv
Duplicate column: index for groups of duplicates.
In our example, there were two groups of duplicates in the input file.
Count column: the index of a duplicate sequence in the group of duplicates it belongs to.
In our example, group 1 had two duplicate sequences – Duplicate1A and Duplicate1B – both of which are indicated in the Sequence column, followed by their identifier in the next column. Group 2 had three duplicate sequences – Duplicate2A, Duplicate2B and Duplicate2C – which are the three sequences indicated.
Sequence column: the duplicate sequences in ascending order (for easy visual checking).
Identifier column: the number from the identifier column in the .csv file or the identifier number after '>' in the .fas and .txt files

Output file 2: SampleUnique.fas
This file contains the set of unique sequences from the input dataset in fasta format. The set of unique sequences includes one representative from each set of duplicates, usually the first one in the set of duplicates.
In our example, we have 3 unique sequences – Unique1, Unique2 and Unique3 – which are present in the output file. In addition, the output also contains one representative from each set of duplicates – Duplicate1A from the first set and Duplicate2A from the second set.

To check if your system has JRE:

Go to command prompt and type java –version.

If your system has java, you will see the version details similar to one shown:

a) If your system does not have java installed, you get the error:
‘java’ is not recognized as an internal or external command, operable program or batch file.
b) You can then download the JRE for your OS from Sun Microsystems: http://www.java.com/en/download/manual.jsp)

=UPDATE=

This program is only able to remove exact EQUAL LENGTH duplicate sequences; it does not treat subset duplicates as duplicates. For example, let's say we have the following sequences:

AAAAAAAAA

AAAAAAAA

The output of unique sequences from the program are:

AAAAAAAAA

AAAAAAAA

You can see that though >2 is a subset duplicate of >1, the program still considers it as unique because it only removes exact same length duplicate sequences, such as >2 and >3, where one was removed and the other kept.

What happens if you have gaps in your input sequences? Let's say we have the following input sequences:

AAAAAAAAA

AAAAAAAA-

AAAAAAAAAB

AAAAAAAAAC

The output that you get from the program is:

AAAAAAAA-

AAAAAAAAA

AAAAAAAAAB

AAAAAAAAAC

Just like in the previous example, only exact equal length duplicates are removed and the gap is treated like an amino acid. So, if you do not want gaps to affect the removal of duplicates (especially if they are in the middle of the sequences), you should remove them using the simple replace function in most text editors (replace "-" with nothing).

So what if you are interested in removing subset duplicates? What program can you use?

You can use Jalview and a tutorial is available here.

Content by: Rashmi & Asif M. Khan
Posted by: Rashmi
Edited by: Asif M. Khan

31) Multiple sequence titles in the same BLAST hit

2009-08-20T16:17:00.014+08:00

We were curious about what it means when BLAST returns a BLAST hit indicated by a “>” but followed by multiple entries with different accession numbers.

Are these sequences the same? And are they the same only at the aligned region or in the rest of the sequence as well?

I did a check on how these sequences are related. Based on several examples (two listed below), for now, it could be concluded that these entries that fall under the same BLAST hit are full sequence duplicates. However, in terms of annotations they could be from different isolates.

Case 1: 2 accession numbers under the same BLAST hit

Alignment between full sequences of Q6R325.1 and AAS01728.1

100% identity, i.e. full sequences are exactly the same
Same author
Swissprot entry (Q6R325.1) has cross-reference to Genbank entry (AAS01728.1)

Case 2: Multiple accession numbers under the same BLAST hit

Multiple alignment of full sequences of all sequence titles

Perfect alignment
Same author
Only the isolate annotation is different

30) Using VLOOKUP (or HLOOKUP) in Microsoft Excel

2009-08-19T13:20:00.011+08:00

VLOOKUP is a very useful formula in Excel to perform tasks such as:

Data comparions between sheets
Checking for discrepancies
Linking data between sheets

and the list can go on!

The formula looks like this:
VLOOKUP(lookup_value,table_array,col_index_num,range_lookup)

--
lookup_value: The value to search in the first column of the table array

table_array: Two or more columns of data. The values in the first column of table_array are the values searched by lookup_value. These values can be text, numbers, or logical values. Uppercase and lowercase text is equivalent.

col_index_num: The column number in table_array from which the matching value must be returned. A col_index_num of 1 returns the value in the first column in table_array; a col_index_num of 2 returns the value in the second column in table_array, and so on. If col_index_num is:

Less than 1, VLOOKUP returns the #VALUE! error value.
Greater than the number of columns in table_array, VLOOKUP returns the #REF! error value.

range_lookup: A logical value that specifies whether you want VLOOKUP to find an exact match or an approximate match:

If FALSE, VLOOKUP will only find an exact match. In this case, the values in the first column of table_array do not need to be sorted. If there are two or more values in the first column of table_array that match the lookup_value, the first value found is used. If an exact match is not found, the error value #N/A is returned.

If TRUE or omitted, an exact or approximate match is returned. If an exact match is not found, the next largest value that is less than lookup_value is returned.

--

The values in the first column of table_array must be placed in ascending sort order; otherwise, VLOOKUP may not give the correct value. You can put the values in ascending order by choosing the Sort command from the Data menu and selecting Ascending. For more information, see Default sort orders.

A good reference for more details of the formula can be found here.
These details are illustrated in the following example.

Step 1:
a) Once you have your table_array (i.e. your “reference dataset” with the first column containing), and the list which you would like to lookup for, enter the VLOOKUP formula in the cell as follows:

b) Note that in the formula, I have put in a “$” sign for the data source table indexes. This is to lock the table_array as A2:B5 and not allow it to slide as we drag the formula down to the rest of the cells.
This is what it’ll look like if I had not put the “$” sign there:

Notice that the data source table_array indexes have shifted (green box) and hence the value “1” could not be found. This will result in a #N/A error, as follows:

To avoid this, it is safer to use the “$” sign in your formula as shown in the Step 1(a).

Step 2: Hover on the edge of the cell, till your cursor becomes a “+”.
Click and drag the formula down to fill the rest of the cells.

Step 3: And this is what you’ll get:

For data comparions from different sheets:
If your source/reference data is stored in another sheet, simply add the sheet name at the beginning of the [table_array] field, like this:

What if I have a horizontal set of data as my table_array?
The VLOOKUP formula described above is used when the table_array is vertical and the lookup values are in the first column.
Use HLOOKUP when the table_array is horizontal and the lookup values are in the top row. The other details, such as using the “$” sign, remain the same.

What does #N/A mean?
The error #N/A indicates that the source for a particular data was not found in the table_array. This could mean either (i) your table_array had shifted, as explained above in Step 1(b) or (ii) the data of interest is not present in the reference table.

Counting the #N/A's
Sometimes we might be interested in the number of data entries that do not have a match in the table_array (the reference table).

To count the number of #N/A errors in a dataset, use this formula:

=IF(ISNA(A1),1,0)

This prints 1 if cell "A1" contains #N/A, else prints 0. Sum up the column to obtain a total.

29) Removing sequences that are fully gapped from alignment using BioEdit

2009-08-19T12:35:00.013+08:00

Let’s say you have an alignment that looks like this after you cleaved out a particular region from a longer protein:
We intend to remove sequences that are fully gapped, as shown in red box above.

To do this,

Click on: Sequence >> Filter out sequences containing certain characters >> Cut them to new alignment

Select Contain non- and type “-“ in the textbox.
This means we are asking the tool to extract any sequence that contains a non-gap character.

Save the output as a new .aln file, and you’ll have an alignment file without the fully gapped sequences!

28) How to import tab delimited data into a table of a mySQL database in proline server???

2009-07-29T13:07:00.005+08:00

Steps to import a tab delimited data:

1) Prepare your tab delimited data. Make sure that there are no headers included on the first line. File transfer in the data to proline either by WinSCP or SSH.

2) Create a table in the mySQL database in proline. Make sure that the imported data would match to the column headers that you have defined in the table respectively.

For example, "D1_00001" in my data would match to "Accession" column that I have defined in my SQL table.

3) Login to proline and SQL. (refer to previous posts 2 and 23)

4) Use the following SQL command:

LOAD DATA LOCAL INFILE 'file_with_full_pathname' INTO TABLE name_of_table

For example:

mysql> \g LOAD DATA LOCAL INFILE '/projects/denvdb/www/DENV.txt' INTO TABLE DENV

It will work even though I did not specify that the data is tab separated.

Please refer here for more information.

For comma delimited data, the sql command is the same except that you may need to add in FIELDS TERMINATED BY ','

However I have not tested this out yet.

27) How to create a metadata file for AVANA???

2009-06-10T17:15:00.006+08:00

In order to do mutual information analysis (e.g in post 25), we need to create a metadata file:

a) Create a blank excel sheet.

b) Paste your GI numbers correspondingly to the first column of the sheet.

c) In the second column, insert the serotype matching with the corresponding gi numbers.

d) In the third column, insert the protein name matching with the corresponding gi numbers.

e) Make sure that you include headers in the first row or AVANA will treat the first data as a header, thus missing out 1 data in your analysis.

f) Once all the data is in, save the spreadsheet as a comma-separated file by changing "Save as type" to "CSV (Comma delimited)".

This is a sample metadata of my dengue virus records with capsid protein entries for all serotypes, which i named as "All_C.csv". The pictures show the start and the end of the sheet respectively.

26) How to convert an alignment file (.aln) to a taln file (.taln)???

2009-06-10T17:03:00.003+08:00

It is important to note that the Antigenic Variability Analysis tool (AVANA) do not recognise a normal alignment file (.aln), and we need to convert it to a .taln format in order to do entropy or mutual information analysis. These are the steps to do it:

a) Open the aln file in BioEdit. Make sure that the misalignments have been corrected.

b) Click File\Export\tab-delimited text (title.tab.sequence)

c) Change "Save as type" as "All Types", and name the file. Make sure to add ".taln" at the end of the filename, and save it to anywhere you want in your desktop.

25) Steps for obtaining mutual information between two sequence alignments in AVANA:

2009-06-05T16:54:00.003+08:00

Steps for obtaining mutual information between two sequence alignments in AVANA: (e.g comparing D1_C to D2_C,D3_C,D4_C):

1) Load your taln file (e.g. All_C.taln) in AVANA

2) Load your metadata (e.g. All_C.csv) in AVANA

3) Go to Alignment\Create Subset

4) In the window, Source Alignment: Master Alignment, choose DENV1 as serotype and C as protein, and give a name to the subset (e.g. DENV1_C)

5) Create the next subset by repeating step 4, but this time highlight the rest (DENV2,DENV3 and DENV4) as serotype, and give another name to the subset (e.g.DENV234_C)

6) Check that the sample size is 9 by going to Tools\Preferences. It will scan for peptides of length 9 which is the typical length of t-cell epitopes.

7) In the same window, under the "Comparisons" tab, tick "Estimate average mutual information" and set the MI threshold to 1.000 (ideally we want value of 1, if get nothing then we consider other thresholds).

8) Next, go to View\Compare two alignments.

9) Click DENV1_C as top alignment and DENV234_C as bottom alignment

10) You will see two graphs. Go to View\Show Mutual Information and View\Show sequence logo.

11) The green line in the graph shows the mutual information. MI value tells u what is characteristic in one alignment when compared to the other alignment. MI value of 1 means, it is seen in this alignment, but was not seen in the other alignment.

12) Click on the position numbers to your left to see the sequence logo, which shows conservation and all the peptides at that position.

13) Export your results by going to View\Statistics\Diversity and save as .txt. Create an excel sheet for better viewing. Sort them according to descending MI, then increasing entropy.

24) Summary steps in activating LAN connection in VMWare if problems still occur after post 22

2009-06-02T12:03:00.004+08:00

Comment by Jean:

Summary steps in activating LAN connection in VMWare if there are still problems in connecting online (after post 22):

a) lspci -vvv and ifconfig -a (display output below, checking what hardware the VMplayer is running from)


    02:01.0 Multimedia audio controller: Ensoniq ES1371 [AudioPCI-97] (rev 02)
            Subsystem: Ensoniq Creative Sound Blaster AudioPCI64V, AudioPCI128
            Control: I/O+ Mem+ BusMaster+ SpecCycle- MemWINV- VGASnoop- ParErr- Stepping- SERR- FastB2B- DisINTx-
            Status: Cap- 66MHz- UDF- FastB2B+ ParErr- DEVSEL=medium >TAbort- TAbort- SERR-             Latency: 64 (1500ns min, 63750ns max)
            Interrupt: pin A routed to IRQ 19
            Region 0: I/O ports at 2080 [size=64]
            Kernel driver in use: ENS1371
            Kernel modules: snd-ens1371

    02:02.0 USB Controller: VMware Inc Abstract USB2 EHCI Controller (prog-if 20 [EHCI])
            Subsystem: VMware Inc Abstract USB2 EHCI Controller
            Control: I/O- Mem+ BusMaster+ SpecCycle- MemWINV- VGASnoop- ParErr- Stepping- SERR- FastB2B- DisINTx-
            Status: Cap- 66MHz- UDF- FastB2B- ParErr- DEVSEL=fast >TAbort- TAbort- SERR- PERR- INTx-
            Latency: 64 (1500ns min, 63750ns max)
            Interrupt: pin A routed to IRQ 16
            Region 0: Memory at e8900000 (32-bit, non-prefetchable) [size=4K]
            Kernel driver in use: ehci_hcd

    eth1      Link encap:Ethernet  HWaddr 00:0c:29:bf:37:04
              BROADCAST MULTICAST  MTU:1500  Metric:1
              RX packets:0 errors:0 dropped:0 overruns:0 frame:0
              TX packets:0 errors:0 dropped:0 overruns:0 carrier:0
              collisions:0 txqueuelen:1000
              RX bytes:0 (0.0 B)  TX bytes:0 (0.0 B)
              Interrupt:18 Base address:0x2024

    lo        Link encap:Local Loopback
              inet addr:127.0.0.1  Mask:255.0.0.0
              UP LOOPBACK RUNNING  MTU:16436  Metric:1
              RX packets:210 errors:0 dropped:0 overruns:0 frame:0
              TX packets:210 errors:0 dropped:0 overruns:0 carrier:0
              collisions:0 txqueuelen:0
              RX bytes:424561 (414.6 KiB)  TX bytes:424561 (414.6 KiB)

b) dhcpcd -k eth1 (kill eth1)

c) ifconfig eth1 up (restart eth1)

d) dhcpcd -nd eth1 (do dhcp call and look for packet advertising the network server from the host, and if found, it will assign an IP address to eth1.)

e) ifconfig eth1 (check that eth1 has an ip now)

23) How to use MySQL in Proline???

2009-06-02T11:09:00.012+08:00

This is a simple tutorial on how to use mySQL commands in Proline.

a) Firstly, log in to the server using putty.exe in Windows or SSH in BioSLAX using your username and password. (See post 2 if you have forgotten how to login through BioSLAX)

b) Next, log in to mysql (it may be a different username and password). See the screenshot below. You will directly enter the database denvdb.

c) Before proceeding, type "\h"(help) to take note of important commands required for navigation through mySQL. See the screenshot below:

Some important commands are:

1) \g, \G : type either of these commands to send an SQL query to the server. \G displays results vertically.

Example: \g SELECT * FROM DENV (display all data in the table DENV). Note that you need to enter the same command twice. See the screenshot below:

2) \c : Clear cache. After an SQL query, use this command if you want to enter another query.

3) \q: Quit mySQL to log back to proline.

Notes:

For uploading a table with data to mySQL, you can use the method on post 4. Otherwise, you can create an empty table with just your fields in the columns using the CREATE keyword. Note that there are many keywords to use in mySQL. You can refer to here for more tutorials on mySQL.

Two keywords that may be important are:

a) Syntax for changing the data types of a column:

ALTER TABLE table_name MODIFY column_name type

Example: \g ALTER TABLE Registration MODIFY contactno INT(10)

b) Syntax for showing column fields, data type, field attributes, primary key and other information in a table:

SHOW columns from table_name

Example: \g SHOW columns from DENV

22) How to fix your internet connection in BioSLAX VMWare

2009-05-19T21:29:00.006+08:00

This tutorial is for those who when after booting up their BioSLAX VMWare find that the internet is not working. For example, you ran Firefox and could not access any website, or ping any site through the Konsole, or could not get the "Wireless Assistant client" to assist you (it returned "No usable wireless device found, wireless, Wireless Assistant will quit now"). You can try the steps listed below to solve this problem:

1. Select the "Bridged" network adaptor (select from the Devices menu on top of the screen) if you are using wired internet. For wireless internet, use the "NAT" adaptor, which forces the virtual machine to talk to the network address card of the host machine, so that the host acts as a router.

2. Type the following in the Konsole to check if your dhcp client is up and running


ps -ef | grep dhcp

# This check the process (ps) number of the dhcp, and if it is running it will be shown as one of the processes. If you have two or more dhcp processes, choose the one corresponding to eth1 if wireless network is desired or eth2 for wired network.

3. If the dhcp is running, kill it to reset things. For example,


kill -9 4663

# Assuming 4663 is the process number of the eth1 dhcp that is to be terminated.

Note: You can kill dhcp and bring down the interface for various interfaces using "dhcpcd -k ", e.g.


dhcpcd -k eth1 or dhcpcd -k wlan0

There is no need to do ps -ef and killing of the process.

4. After killing the dhcp, you want to check whether the eth1 or 2 card is being recognized by BioSLAX. Type


ifconfig -a

# If the interface card (eth1 or 2) is listed, along with the loop back, this means that the eth1 (or 2) card is recognized by Bioslax. Now, to check whether the eth1 (or 2) card is up and running, type


ifconfig

# If you only see loop back, then it means that your eth1 (or 2) card is not running. You need to start the eth1 (or 2) by typing


ifconfig eth1 up

# Change eth1 to 2 if you want to up eth2
# If desired, you can bring eth down in the same manner

5. Once the eth1 (or 2) is up and running. You need to start the dhcp. Type


dhcpcd -nd eth1

# This will do dhcp call and look for packet advertising the network server from the host, and if found, it will assign an IP address to eth1. When successful, the connection established will pop up, courtesy of the BioSLAX GUI.
# For eth2, change eth1 to 2

You are done!

Summary code


1. dhcpcd -k eth1
2. ifconfig eth1 up; dhcpcd -nd eth1

Note
If you are not running bioslax with persistence change saving mode, you will need to redo this step every time you reboot BioSLAX.

21) How to do multiple sequence alignment using MUSCLE

2009-05-13T15:21:00.006+08:00

MUSCLE is an alternative to ClustalX or W to construct a multiple sequence alignment (MSA). MUSCLE is particularly good at handling large number of sequences for MSA and produces better quality alignments when numerous short partial sequences are present in the input fasta file. Below are the steps to run MUSCLE alignment using the command prompt:

1. Create a new folder on your computer and download MUSCLE to this folder from

http://www.drive5.com/muscle/downloads3.6/muscle3.6_win32.zip

2. In the same folder paste a copy of your input sequences to be aligned.

3. Now, follow the instructions below to run MUSCLE using the command prompt (Start-> All Programs -> Accessories -> Command Prompt; note that the procedures below for the command prompt are all case-sensitive, so make sure you type carefully!)

4. Now, navigate the command prompt to the folder you just created for MUSCLE alignment. Here are some tips to help you move around in the command prompt:

- to go one folder level backward: cd..
- to list content of director/folder: dir
- to go one folder level forward: cd

5. Once you have navigated to the folder, type “muscle –in –out ” to perform the alignment.

6. Close the command prompt when the alignment is complete.

Note: The output file is in “afa” format, which is not suitable for subsequent analyses by other programs. Therefore, you should convert it to the popular “aln” format. To do this, load the output file into ClustalX and then click on File -> Save Sequence as -> under format, select “Clustal format” -> under “Save Sequence As” at the bottom, change the name of the file extension from “output_file.afa” to “output_file.aln” -> click on the OK button.

Content by: Sye Bee & Asif M. Khan
Posted by: Sye Bee
Edited by: Asif M. Khan

20) How to use Blastclust to identify representative sequences

2009-05-13T12:48:00.012+08:00

A situation where this may be applicable is when you have too many sequences and you want to do a quick analysis by using a set of sequences that represent all the sequences that you have. For example, you have a dataset of 1000 sequences of length 600 amino acids each and you would like to do phylogenetic analysis on them using Neighbor joining algorithm. However, because of the large input dataset, it will be few days before you get your results. So, in this case, you could choose to just analyse sequences that represent all your 1000 sequences. Follow the steps below to get your representative sequences:

1. Go to http://toolkit.tuebingen.mpg.de/blastclust

2. Load your input sequence file and set the length to be covered to 100%, and the percent identity to 90% or to any other desired threshold.

3. You may either wait for the results to appear or wait for an email about the results from the server

4. Here is an example of the results

5. Save the resulting output (representative sequence fasta file), by clicking on the “save” button on the "Results" bar, and save your file with a “.Fasta” extension.

6. Check the unique representative fasta file by opening it in a text editor.
The file may look weird in Notepad, use Wordpad instead or get a good text editor,
such as Editplus or Notepad++, among others. You are done!

Content by: Asif M. Khan & Sye Bee
Posted by: Sye Bee
Edited by: Asif M. Khan

19) How to remove duplicate sequences from a fasta formatted input file

2009-05-13T12:31:00.013+08:00

===Update: 21 March 2011===

For a more comprehensive and updated information on this, please see Post 32.
Read all the way to the end of that post.

======================

You can use Jalview to easily check for duplicates and remove them if any.

1. Download and install Jalview on your home system from

http://www.jalview.org/download.html

2. Run Jalview and close all example windows

3. Load your fasta file to Jalview

4. Remove duplicates:

- Select all sequence

- Go ‘Edit’ to uncheck the pad gaps function.

- In ‘Edit’, select ‘Remove all gaps’

- After that select ‘Remove redundancy’

- At the “redundancy threshold selection” dialog box, set the threshold value to 100, click ‘Remove’.

5. Saving the unique fasta file and you are done!

Content by: Asif M. Khan & Sye Bee
Posted by: Sye Bee
Edited by: Asif M. Khan

18) How to shorten the description line in fasta format to just include GI number

2009-05-13T12:16:00.014+08:00

Some of the software like Phylip or Clustal (version 1.6) truncate the description line and only take the first 10 characters. This may result in duplicate description lines in your input sequence file. You could choose to just have the GI number in the description in order to avoid this problem as it has a maximum of only 9 characters. Follow the steps below to achieve this:

1. Open the Fasta formatted input file in MS Excel

2. Select column A and click on “Ctrl+H” for the replace function

3. You have to replace “gi|” with nothing, follow the steps below:

a. Under “Find what”, type “gi|”, and leave “Replace with” blank

b. Click on “Replace All”

You will see this upon successful replacement:

4. Next, replace everything after the “|” symbol with nothing:

a. Under “Find what”, type “|*”, and leave “Replace with” blank

b. Click on “Replace All”

Output upon successful replacement. Now you only have the GI numbers left:

5. Save the file by clicking on the Save icon or “File” à “Save”

6. Click on “Yes” when the warning prompt pops-up

7. Close Excel

8. Ignore the prompt that pops-up by clicking on “No”

9. Open your fasta file in Notepad to check whether only the GIs are left on the description line, if yes, you are done!

Content by: Asif M. Khan & Sye Bee
Posted by: Sye Bee
Edited by: Asif M. Khan

17) Installing BioSLAX as a full Linux system to your hard drive

2009-05-11T15:59:00.017+08:00

Follow the instructions below to install BioSLAX (see post 9) as the second Operating System (OS) on a system preinstalled with Windows OS and to configure the dual boot option when booting the computer. We do not guarantee that the steps below will work fine on your system, though we had no problem implementing them on ACER Extensa 4620. So, follow the steps below at your own risk.

1) Boot up with the latest BioSLAX DVD with all the fixes.

2) When it boots up into KDE, from the KDE menu, under system, you should find the "GParted Partition Editor".

3) Use the partition editor to unmount the hard disk that you want to resize (right click -> Unmount) (Figure 1). Select “Do Nothing” when a KDE daemon pops up. Drag the hard disk to resize the partition, so that there is at least 4 Gb (about 4,000 MiB) of free hard disk space for the installation of Bioslax (Figure 2 & 3). Right click on the empty partition created and click “New” (Figure 4). Then, format the new hard disk partition to ext3 (Figure 5). Ignore the KDE daemon that pops up asking if you want to mount it again - click "Cancel".

Figure 1: Unmounting /dev/sda2/

Figure 2: Resizing /dev/sda2/

Figure 3: Defining the new size of /dev/sda2

Figure 4: Creating a new partition in your hard disk

Figure 5: Formatting the new partition as ext3

Figure 6: Applying changes to the hard disk

4) Click on “Apply” at the top bar of the partition editor to apply your changes (Figure 6). This may take 2 – 3 hours.

5) If the filesystem indicates that the new partition is unformatted, right click on the empty partition and format it to ext3.

6) Re-mount the hard disk by right clicking and selecting "Mount on" (should be mounted to /mnt/sdaX or /mnt/hdaX depending on whether it’s a sata or ide disk). If there is no selection for mounting, close and reopen Gparted Partition Editor, and try again.

7) Go to the KDE menu, under BioSLAX for the BioSlax installer (Figure 7).

Figure 7: BioSLAX Installer

8) Leave the "Source" as whatever it shows (can be blank), the "Install BioSLAX to" should be the partition you formatted and mounted earlier in step (4), "Write MBR to" should be /dev/sda or /dev/hda depending on the type of disk you formatted ealier (sata or ide).

9) Click “Install” and wait for it to finish. This may take 10 – 15 minutes.

10) Reboot and you should see the lilo screen for about 2 seconds before it boots into Bioslax. Login with username: root and password: bioslax. Type “startx” in the command screen to switch from command mode to graphical mode, “reboot” to restart the computer and “init 0” to shut down the computer.

Figure 8: Locating /etc/lilo.conf

11) To enable dual booting at the lilo screen, boot up Bioslax and add these lines to the end of the /etc/lilo.conf (Figure 8):
# windows
other = /dev/hdaX (or /dev/sdaX if it’s a sata disk)
label = Vista
where hdaX is the partition where windows is.

Figure 9: Running /sbin/lilo on Konsole

12) After that, start up Konsole and run /sbin/lilo to reread the config and reboot (Figure 9). You should see 2 choices there for about 2 seconds. Change the timeout in /etc/lilo.conf from 20 to 50 or 100 to get 5 or 10 second delays instead of just 2 seconds. If you want it to default boot to Vista instead of BioSlax then add the following line after the "vga=.." line :
default=Vista
Note: Whatever changes you make to the /etc/lilo.conf, make sure to run /sbin/lilo to reread them.

Content by: Jean, Benjamin, Mark and Kuan Siong
Posted by: Sye Bee & Asif M. Khan

16) LAPIS: Fasta formatting a list of sequences

2009-05-09T15:15:00.005+08:00

Let's say you have a list of sequences (one sequence per line) downloaded from a databases and you would like to FASTA format the list. To quickly do this, follow the steps below.

1. Copy and paste the list to an excel sheet
2. Insert a new column before the sequence list column
3. In the new column enter number 1 in cell 1 and number 2 in cell 2, and then to auto fill the numbers in the rest of the cells, you highlight cell 1 and 2 and click-drag the right bottom corner of the second cell to the last cell of the list
4. Copy the two columns to LAPIS as a new file
5. Activate the simultaneous editing mode
6. On the first line, click in between the description portion and the sequence portion of the line. E.g. place and click the cursor after the number "1", which is just before the sequence
7. Hit the enter button. This will move the sequence portion below the description portion in a new line. Thanks to the simultaneous mode, this will be automatically done for the rest of the sequences
8. There might be extra leading spaces before the sequence. So, to remove them, click at the beginning of any one of the lines containing the sequence. Then, click on the delete button until all the leading spaces are gone. Thanks to the simultaneous editing mode, all the leading spaces in all the other sequences will also be removed.
9. Save the file as a text file. You are done

Posted by: Asif Khan

15) General steps that you need to do after transferring the webpages from localhost to another server

2009-05-08T11:53:00.003+08:00

These are steps that you need to do after transferring your webpages (e.g. DENVDB webpages) from localhost to another server (e.g. proline):

a) Edit the config.inc.php (change the username, password and database name if required, dbhost remains the same [localhost])
b) Export the DENV table from localhost SQL and import into proline SQL (using post 4 of bioinfotutlets)
c) Setup wwwblast in Proline (using post 7 of bioinfotutlets)

14) How to convert a phylip file to an alignment file

2009-05-08T11:45:00.003+08:00

To convert a phylip file to an alignment file:

a) Open the phylip file in BioEdit
b) Go to File\Export\Sequence Alignment
c) Select as type: ClustalW 1.8 (*.aln), name your file and save!!!
d) Since this approach does not provide the sequence conservation line (*, : or .) in the aln file generated, you re-save the file in the aln format using ClustaX: i)Open the aln file from c in ClustaX, ii) File --> Save Sequence as --> Select "CLUSTAL format" under format --> Click on the "OK" button. Now you should be able to see the sequence conservation line.

13) How to create a bootable (e.g. BioSLAX) USB thumbdrive

2009-05-08T03:58:00.001+08:00

First, format your USB thumbdrive as a FAT or FAT32 filesystem using your Windows format utility. Note that the thumbdrive you use must be of capacity 1 GB and above.

Using any software that can extract files from an ISO (eg. Magic ISO, WinISO or WinRAR), extract the contents of the operating system (OS, such as BioSLAX) ISO into your USB thumbdrive. Alternatively, you can copy and paste the boot and bioslax folders from your Bioslax Live DVD/CD to your USB thumbdrive directly. You will find 2 directories in your thumbdrive, namely boot and bioslax.

Using a DOS command prompt (Windows Start button, click Run, then type in “cmd” without the quotes into the textbox and hit enter), CD to the boot directory. Now run the file called bootinst.bat. (You can also double click on bootinst.bat in your USB directory in file explorer). It will show you the following text :

Where %DISK% is the drive letter of your USB thumbdrive. Hit any key make your USB thumbdrive bootable.

IMPORTANT! A bootable USB thumbdrive will only work if the machine you are booting from supports booting from external USB devices. If it does not, the machine will not recognize the thumbdrive as bootable and will not boot BioSLAX from it.

Content by: Mark De Silva, Lim Kuan Siong, Tan Tin Wee

Posted by : Sye Bee & Asif M. Khan

Edited by : Sye Bee & Asif M. Khan

12) How to make a backup of Live CD/DVD

2009-05-08T03:57:00.000+08:00

Once you have created your Live CD/DVD, it is highly recommended that you make a backup of the CD, follow the instructions below:

1 Run the CDBurnerXP Program, switch to the “Copy disc” option and click on the “Copy Data” button.

2 Insert the BioSLAX Live CD/DVD into your driver, select the options as shown and then click on the “Copy disc” button.

3 Insert a blank CD/DVD-R to your CD/DVD R/W drive when this message prompted.

4 The backup process is completed when you see this message:

Content by: Asif M. Khan

Posted by : Sye Bee & Asif M. Khan

Edited by : Sye Bee & Asif M. Khan

11) How to burn an ISO file (e.g. BioSlax.iso) into a bootable Live CD/DVD

2009-05-08T03:55:00.001+08:00

To burn an ISO file (such as BioSlax.iso) into a bootable Live CD/DVD , you should use a CD/DVD burning program with an image file creating/editing/extracting capability, such as CDBurnerXP, which is available for free at http://cdburnerxp.se/ and works on Windows 2000/XP/2003/Vista.

Follow these step by step instructions to burn an iso file:

1 Download and install the CDBurnerXP program from http://cdburnerxp.se/.

2 Run CDBurnerXP Program, change to “Burn ISO image” option, click “OK”.

3 Insert a blank CD-R/DVD-R to your CD/DVD R/W drive, load the ISO image file. Select the options as shown below then click on the “Burn disc” button.

4 You should see this once if the writing process get started.

5 if you see a message as below, you have successfully created a copy of the BioSLAX Live CD/DVD from the image that you downloaded!

Content by: Asif M. Khan

Posted by : Sye Bee & Asif M. Khan

Edited by : Sye Bee & Asif M. Khan

10) How to use/run the BioSlax Live CD

2009-05-08T03:24:00.001+08:00

BioSlax is a new Live CD operating system, developed in our lab, as a comprehensive system for Bioinformatics workstation client, server resource and teaching platform. It has thus far been successfully used for teaching and training by several educational institutes, including the National University of Singapore where we use it to teach our advance bioinformatics course (LSM3241) students. Watch the YouTube video below for a demo on how to use the Live CD (this video, btw, happens to be my first YouTube video, so, please pardon the amateurishness; the voice is mine and the hand gestures are by my colleague Ms. Hu Yongli). Visit www.bioslax.com for more information (i.e. documentation) and to download a copy of the latest ISO image file.

Posted by: Asif Khan

9) A Guide to Using BioSLAX

2009-05-07T18:17:00.023+08:00

BioSLAX is a new live CD/DVD operating system created to facilitate bioinformatics software usage and has been used for teaching and training by several educational institutes. Bootable from any PC, this CD runs the compressed SLACKWARE flavour of the LINUX operating system also known as SLAX.

Distribution of the Live CD/DVD systems as bootable CDs or their images (~700MB), is free to educational institutions and so far has been done via http, ftp, bittorrent, p2p and physical CD. With portable and scalable systems like BioSLAX, it is now easy to set-up bioinformatics labs, fully equipped with LAMP (Linux-Apache-MySQL-PHP/PERL), Mediawiki and more than 200 freely available bioinformatics software, see below. New software packages can always be made available by remastering the live CD/DVD systems.

Software preinstalled in BioSLAX
° Mozilla Firefox (web browser)
° Open Office
° BLAST
° ClustalW
° ClustalX
° EMBOSS
° Modeller
° PamL
° PatScan
° T-Coffee
° PHYLIP
° GeneSplicer
° GlimmerHMM
° HMMER
° Primer3
° Artemis ACT
° Artemis ART
° jAlign
° Jalview
° NjPlot
° PyMOL
° RasMOL
° ReadSeq
° TreeView
° Sequence Manipulation Suite v2

1. Getting a copy of BioSLAX

There are several ways of booting BioSLAX, but all of them will involve the initial step of obtaining the BioSLAX iso image (a kind of special file) and burning it into a bootable DVD/CD or thumbdrive. You can download the latest BioSLAX iso from either one of the following URLs:

ftp://sf01.bic.nus.edu.sg/incoming/bioslax/distro/v7.1/
http://www.bioslax.com/download/bslax71-dvd-incob.iso

Note that when downloading using a Web browser, you have to turn off your cache, or set the cache to zero to prevent a cache copy from being created. In this way, the iso file will immediately be written to your hard disk. Save it conveniently as C:\slax\bslax71-dvd-incob.iso or something easier to type, e.g. C:\slax\bslax.iso.

2. Creating a bootable DVD/CD or USB thumbdrive

Once you get the iso copy of the BioSLAX, you need to save it into a DVD/CD or a USB thumdrive, favorably, please make a backup copy as well.

*Note that using any software that allows burning of DVDs/CDs from iso files (eg, Nero, EZ media creator, Magic ISO, or CDBurnerXP) enable you to burn the downloaded iso file into a bootable DVD/CD, this is NOT the same as copying the iso file into the DVD. Burning the image means transferring the entire image of the iso file including the file system and all its constituent files into the DVD/CD, instead of copying the iso file into the DVD/CD’s file system.

Please refer to the “How to burn the ISO into a bootable Live CD/DVD”, “How to create a bootable USB thumbdrive” and “How to make a backup of the Live CD/DVD” sections if you have no idea on how to do these.

3. Starting BioSLAX by booting from the Live DVD/CD or USB thumbdrive

Method 1: Booting directly from the CD/DVD/USB thumbdrive

There are two ways of doing this:

a. Selecting CD/DVDROM or USB device as the boot device when starting the computer.

When you switch on the computer, you will see a line of instructions asking you to press F12 (or some other key depending on the PC) for you to select Boot Menu. Press it, then select to boot from the CD/DVD Drive or the USB device.

Typically, the options may look like as follows:

Boot Menu
Floppy Disk
USB Device
Hard Disk
CD/DVD Rom
Network

When you see this screen, use the arrow keys to move up and select the CD/DVDROM option (if you are booting from DVD) or the USB DEVICE option (if you are booting from a USB thumbdrive), and press ENTER. The boot sequence will then read from the CD/DVD Rom or USB thumbdrive, where BioSLAX is located.

If this doesn’t work, try option b. If it works, skip the next step (b).

b. Configuring your Boot Device Priority of the BIOS of your PC.

When you boot up your computer, press F2 (or ESC, or DELETE, etc, depending on your PC) to enter into the BIOS Configuration mode. Select the BOOT tab, and the BOOT device priority list. Select CD/DVDROM or USB Device to be the first boot device by using the arrow key or PgUp depending on your PC. Save and Exit (typically pressing F10).

When this is configured properly, the PC knows to boot from the CD/DVDROM or USB DEVICE first (instead of the usual Hard Disk). BioSLAX will then start to load as the operating system.

Figure 1. BioSLAX booting up

When it does boot up from BioSLAX, you will see the BioSLAX splash screen as in the first diagram in Figure 1.

BioSLAX has several booting modes to cater for different experiences and hardware. For most of the new PCs and laptops, the first option should be fine. If you are having trouble with your display, choose the 2nd option which is the Safe option. If any of your hardware fails to work, try to boot with the other (No ACPI or No PCMCIA) options.

Method 2: Booting from the BioSLAX files on a hard disk

Generally, a Pentium 4 machine with 512 Mbyte RAM is the minimum configuration that should just work. However, you might find that the overall experience of booting from the CD is too slow.

If so, you can make it boot faster, by having the system access the BioSLAX files from your hard disk. Reading data from the hard disk is generally faster than reading data from a Bioslax CD/DVD in the CD/DVDRom Drive.

There are 2 ways to boot from the hard disk,
a. from files of BioSLAX copied into the hard disk
b. from a single BioSLAX.iso file containing the BioSLAX image, copied into the hard disk.

Copying the contents of the CD/DVD/USB thumbdrive onto your hard drive

Boot up as usual into your Microsoft Windows environment. Create a subdirectory on your Windows hard drive (eg; C:\SLAX) then copy the contents of the BioSLAX CD/DVD or the USB thumbdrive to that directory. Just drag and drop the contents into the C:\SLAX directory in your file explorer.

IMPORTANT NOTE:

In the Microsoft Windows (XP or Vista) environment, disk drives are typically labeled C: or D: for hard disks, A: for Floppy and E: for CDROM drives.

Now, in the Linux OS, the different drives are not represented by letters (eg, A, C, D, etc). They are represented by devices. Again these are represented differently for IDE, SATA and SCSI hard disk drives. The following is a table showing the different representation of the different drives:

Therefore C:\SLAX in the Microsoft Windows OS would be represented as /dev/sda1/slax to the Linux OS (if it is a SCSI or SATA drive). Note that Linux is CASE SENSITIVE, so the case of the directory name matters.

Once you have copied the contents of the DVD onto your hard drive or have copied the BioSLAX ISO to your hard drive (eg C:\bslax.iso), you have to use the BioSLAX CD/DVD or USB thumbdrive to boot up, and at the initial BioSLAX menu selection screen, press the TAB KEY while over any of the boot up selections of your choice.

In the following example, we will assume you are on option 2 (Safe option).

i. Press the TAB KEY while the selection bar is over option 2.
ii. You will see a line of text at the bottom.

Figure 2. Select your option and press the TAB KEY

Figure 3. Command text appears at the bottom after pressing the TAB KEY

iii. Press the space bar and then :

A) If you want to boot from the BioSLAX files extracted to your hard drive (files in C:\SLAX), type from=/dev/sda1/SLAX

Figure 4. Booting from BioSLAX files extracted to the hard drive

B) If you want to boot from the ISO file (C:\bslax.iso), type from=/dev/sda1/bslax.iso

Figure 5. Booting from a single BioSLAX ISO file on the hard drive

The system will start to boot up and will read the BioSLAX data from either the directory where the extracted BioSLAX files are or from the BioSLAX ISO file directly.

When the system boots up, you will see the device initialization screen and finally the X-Windows desktop.

Figure 6. BioSLAX device initialization screen and X-Windows desktop

NOTE: When booting from the iso or files from the hard disk, you need a BioSLAX DVD/USB thumbdrive to initiate the boot up, after which it will start to read the files from the hard drive. Since you won't know exactly when it starts to read from the hard drive, it’s best to leave the DVD/USB thumbdrive plugged in throughout the entire BioSLAX session.

TIP: name your iso to something easier to type, like b.iso. All you have to type now isfrom=b.iso

4. How to connect to the Internet

a. Wired connection

Plug your network cable to your PC and BioSLAX will auto detect it immediately and set up your network connections.

Consult your TAs if you are connecting from your home LAN.

b. Wireless connection

Wireless Asst à Insert wireless card à Select eth1.
If wireless network card is built-in, select eth0.

Select DHCP and an error message appears, at the same time as a pop-up at the lower bottom right corner. Run your Mozilla and login to NUSNET WINZONE EZXCESS login page and select NUSSTU, your login id and associated password.

5. Wireless Networking Support

If the above Wireless network connection doesn’t work, please note that BioSLAX has support for a variety of wireless cards and onboard wireless devices running the following chipsets:

° Intel 2100/2200/3945/4965
° Athereos
° TI ACX11x
° Broadcom
… and many more

Most laptops today run on the Intel wireless 3945 or 4956 which is fully supported by BioSLAX.

Note:
WPA/WPA2 secured access points cannot be accessed using the wireless manager. This requires more command line configuration which will not be covered in this document.

6. Saving Changes

After doing your work on BioSLAX you may have made changes to the configuration. Every time you reboot your machine, ANY CHANGES YOU HAVE MADE WILL BE LOST. To prevent this, you need to save changes regularly.

For portable data (eg, documents, results files, etc) you can plug in your USB thumbdrive and save the data to the USB thumbdrive and then access it later even after rebooting, but how do you save system changes?

Firstly, determine what device your thumbdrive is identified as in linux. It may be identified as /mnt/sdb. It could also be sdc or sdd instead of sdb, depending on how many other sata/scsi devices you have on the computer. For this example, we will assume that it is /mnt/sdb.

Make a directory on the thumb drive called mychanges.

When booting up, again hit the TAB KEY when you are at the BioSLAX menu selection. The command text will appear at the bottom. Press the space key and enter the following:

changes=/mnt/sdb/mychanges

Figure 7. Setting up persistent changes

If you are booting from the extracted BioSLAX files or the BioSLAX iso, proceed as instructed in section 3, method 2, and type the changes= command after the from= command.

With this command, any changes or modifications you make will be saved AND restored from the mychanges directory on your thumbdrive.

Note:
When you are saving changes with your BioSLAX booted up from a particular source (USB thumb drive, hard drive etc.), you must reboot BioSLAX from the same source to restore your changes. If you reboot BioSLAX from another source, you won’t be able to restore the changes that you’ve made.

For the more intuitive students, you will realize that you can create different directories on your thumbdrive to save different sessions of your BioSLAX runs. You may set up BioSLAX in different ways or run a sequence search using different programs and store the results individually, so that you may later do a comparison.

Content by: Mark De Silva, Lim Kuan Siong, Tan Tin Wee
Posted by: Sye Bee
Edited by: Sye Bee & Asif M. Khan

8) General steps to do after transferring webpages from localhost to proline:

2009-05-06T14:48:00.002+08:00

General steps to do after transferring webpages (e.g. DENVDB files from htdocs) from localhost to proline:

a) Edit the config.inc.php (change the username, password and database name if required, dbhost remains the same [localhost])
b) Export the DENV table from localhost SQL and import into proline SQL.
c) Setup wwwblast in Proline.

7) Setting up wwwBlast on Proline server

2009-05-04T17:11:00.002+08:00

To setup wwwBlast on proline.bic.nus.edu.sg (at start):

a) In Bioslax, copy the folder containing the formatted blast database (after using formatdb), paste it inside /usr/local/wwwblast-2.2.17, and rename the folder as db.
If there is an earlier db folder in /usr/local, delete it.

b) Copy the whole wwwblast-2.2.17 folder and tar it.

c) Copy the tar file into the directory /home/benjamin in proline.bic.nus.edu.sg using WinSCP. Ultimately you can use the file transfer procedure above if you do not want to leave Bioslax. Login into the proline server using Asif's account, as he has sudo access.

d) Copy the tar file from the directory /home/benjamin into /usr/local in the proline server by using the command:

sudo cp /home/Benjamin/wwwblast-2.2.17.tar.gz /usr/local/wwwblast-2.2.17.tar.gz

The sudo command is used as Asif do not have the complete permissions for the proline server.

e) Untar the tar file (it should be in /usr/local by now!!!) by using the following command:

sudo tar -zxf wwwblast-2.2.17.tar.gz

To setup wwwBlast on proline.bic.nus.edu.sg (with new data):

a) In Bioslax, copy the folder containing the formatted blast database (after using formatdb), paste it inside /usr/local/wwwblast-2.2.17, and rename the folder as db.
If there is an earlier db folder in /usr/local, delete it.

b) Edit the blast.html and rc files (blast.rc, ncbi_blast.rc, psiblast.rc, rpsblast.rc) inside /usr/local/wwwblast-2.2.17 to include the names for the new database.

c) Copy the whole wwwblast-2.2.17 folder and tar it.

d) Copy the tar file into the directory /home/benjamin in proline.bic.nus.edu.sg using WinSCP. Ultimately you can use the file transfer procedure above if you do not want to leave Bioslax. Login into the proline server using Asif's account as he has sudo access.

e) Copy the tar file from the directory /home/benjamin into /usr/local in the proline server by using the command:

sudo cp /home/Benjamin/wwwblast-2.2.17.tar.gz /usr/local/wwwblast-2.2.17.tar.gz

The sudo command is used as Asif do not have the complete permissions for the proline server.

f) Untar the tar file (it should be in /usr/local by now!!!) by using the following command:

sudo tar -zxf wwwblast-2.2.17.tar.gz

6) Using file transfer from local computer in Bioslax to another server

2009-05-04T17:10:00.000+08:00

To use file transfer from local computer in Bioslax to another server (e.g. proline.bic.nus.edu.sg) without leaving Bioslax:

Type the following command in Bioslax Konsole:

scp filename username@servername:/directory_in_server

e.g scp wwwblast-2.2.17.tar.gz benjamin@proline.bic.nus.edu.sg:/home/benjamin

The file wwwblast-2.2.17 would be copied and pasted to the location /home/benjamin in the proline server.

5) Compressing and uncompressing a folder in Bioslax/Linux environment

2009-05-04T17:07:00.001+08:00

To compress a folder in Bioslax/Linux environment:

a) Navigate to the folder using Bioslax Konsole

b) Type the following command:

tar -zcf ____.tar.gz ______

where the first line represents the name of the tar file that you want to name as, and second line represents the folder that you want to compress.

To uncompress a folder in Bioslax/Linux environment:

a) Navigate to the directory you want to uncompress in.

b) Type the following command:

tar -zxf ____.tar.gz

where the first line represents the name of the tar file that you have name it as.

4) Exporting SQL database from Bioslax and importing to another server's SQL database

2009-05-04T17:05:00.003+08:00

To export SQL database from Bioslax and import to another server's SQL database (e.g. proline.bic.nus.edu.sg)

a) Type the following command in Bioslax Konsole:
#mysqldump -u username -ppassword database_name > FILE.sql

e.g #mysqldump -u root -phahahaha DENVDB > FILE.sql

FILE.sql would be generated in /root. Copy the file to Windows Desktop just in case.

b) Type the following command in Putty Konsole or SSH in Bioslax Konsole (server side):
#mysql -u username -ppassword database_name < FILE.sql

e.g #mysql -u benjamin -phahahahah denvdb < FILE.sql

Justin has created the database as denvdb.

Got an error message:
ERROR 1045 (28000): Access denied for user 'benjamin'@'localhost' (using password: YES)

Justin has enabled CREATE and ALTER access for my account, so it works now.

Tips from Justin:

1. don't type your password on to a command line ... people can just see what you are typing by using commands such as finger or who
e.g. instead of typing ...
#mysqldump -u username -ppassword DENVDB > FILE.sql

type
#mysqldump -u username -p DENVDB > FILE.sql

then the system will prompt you for a password and then only you type in there (it won't be shown on the screen as you type, so no worry)
also, by typing your pass on the command line, others can easily just check your ~/.bash_history file to see what you have typed and hence get your pass

2. in Windows, it is not case sensitive since it doesn't have much concept of case sensitivity ... in Linux, UNIXes etc
yes it matters. So, preferably you just use small case for most things (if possible) ... and try not to use spaces in between names as well ...
it will make a lot of things easy .. besides, you need less effort following this simple convention.

3. before you try to do loading and stuffs, you can always check with account login first.
just login to proline and type

mysql -u benjamin -p

this way you can test to see if you can even login or not in the first place ... and if yes, then you can narrow down what went wrong.
Anyway, assuming that you managed to get in as what I've done using ur account, you will see there are two databases ...
one is denvdb the other is information_schema. The latter is a system default so ignore that.

with that, then next thing is ... in your FILE.sql ... do you have a statement like

use denvdb;

right at the top of the file ?
if you don't , then you have to do

mysql -u benjamin -p denvdb < file.sql

3) Generating High Quality Publication Figures

2009-05-04T01:27:00.002+08:00

Methodology

Works for all images in MS Word, Excel and Powerpoint.

1) Copy the image to MS Word. If it is an Excel figure, you can leave it there, unless it does not fit an A4 page (then you move it to MS Word)

2) Convert the document to PDF (using the print option) with standard settings (600 dpi), and make sure the correct page size is selected (usually A4 works). Also, go to layout and click on advance, make sure the dpi setting there matches with that of the pdf pdi setting (in this case, 600dpi). And if you want to print in greyscale, change the colour to black. For excel figures that do not fit A4, you can try page sizes larger than A4 and then later crop the empty spaces.

3) Crop the document to the required size. Go to document menu --> crop --> remove white spaces. If you have a page number at the bottom, make sure you remove it first before you select "remove white pages" option.

4) Export to TIFF. File--> Export-->Image-->TIFF-->Set setting to 600dpi and the relevant colour options --> Save. When prompted, also save the cropped PDF.

5)You are done, you now have your 600dpi image file, which you can insert to a MS Word document to send to the journal.

Dealing with Graffle images

1) Export the image to Photoshop format at 600dpi.

2) Open the image in Photoshop and save it as TIFF@600dpi

OLD METHOD

1) Copy and paste the MS Excel/Powerpoint figure to MS Word (if copying from Excel, paste as enhanced metafile).

2) Adjust the image to fit the desired size. Take note of the size of the final image.

3) Convert the MS Word document to PDF with 600 dpi setting

4) Open the PDF file, set zoom to 100% and then select the image to be converted to TIFF (Tools --> Select & Zoom --> Snapshot Tool). Selecting zoom to 100% is very important because you want to capture the maximum quality of the image at its original size. This step is not so critical for figures exported from the Graffle program (For Mac Only) because when the image is exported, it is already exported to 100% magnification.

5) Copy and paste the image from PDF to Photoshop. File--> New --> don't change any settings yet in the window that pops up, except for the part whether you want the image to be greyscale, RGB or CMYK.

6) To set the image to the desired resolution (for example, 600dpi)using Photoshop: Image --> Image Size - -> Set the resolution to 600dpi. Ensure that you have selected the dpi and not the dpcm option.

7) To set the image to the original size in the MS Word document (see point 2) using Photoshop: Image--> Image Size --> In the "Document Size" section, set the width and the height to the original size of the image in MS Word before it was converted to PDF (see point 2).

8) Save the image as TIFF with LZW compression.

9) As a backup also save the photoshop file in case you want to make changes in the future.

10) Insert the saved TIFF image to a MS Word document and compare its quality with the original image. Don't trust what you see on screen. It is always good to print out the image to check how it looks on paper. Even though, sometimes it is difficult to beat the original image that you generated from MS Word, Excel or Powerpoint, the TIFF images generated are usually of reasonable high quality and file size for publication purposes. Most of the quality is lost when compressing the image to smaller size and we have no choice but to do that to avoid clogging up the disk space on the journal's server.

2) How to connect to remote server using the Konsole in BioSlax?

2009-04-22T15:44:00.002+08:00

To connect to remote server using the Konsole in BioSlax:

For example, if you want to connect to proline.bic.nus.edu.sg:

- Enter Konsole
- Type "ssh username@proline.bic.nus.edu.sg"
- Type your password.
- You should see "username@proline:~$". This indicates that you are connected to the server.
- Change to a new password by typing "passwd", and follow the prompts.

1) How To Automate BLAST Search using Network BLAST?

2009-04-02T00:44:00.007+08:00

===Update: 20 March, 2011===

Question to NCBI:

NCBI will no longer produce newer versions of NetBLAST?

Is the legacy version still supported?

Reply from NCBI:

"NetBLAST was rewritten into blast+ as the "-remote" option, so it will be maintained there. Legacy blast (blastall, blastcl3, etc.) is still being supported, but that will stop at some, unknown point in the future. I'd guess it will be supported for at least another year. That does not mean that we will address all bugs, particularly for blastcl3 (NetBLAST)."

=======================

One can use Blastcl3, the NCBI BLAST network client, to remotely connect to the NCBI BLAST servers to run automated BLAST searches. No web browser is required, you don't have to wait in front of the computer for the results and can perform "batch" search of many sequences against the databases at one time. The results of the search using this Network Blast will be identical to that of the Web version (http://blast.ncbi.nlm.nih.gov/Blast.cgi), as long as the identical parameters are used. I have tried and tested this.

Note on blast parametes for users who will rely on the Web version for the parameters to be used on the Network version:

If you don't uncheck (untick) the option for short sequence queries ("Automatically adjust parameters for short input sequences") on the Web version, the default algorithm parameters that are displayed on the input page will not be used (for example, E-value of 10, Blosum 62 matrix, Word size 2, etc). This I think is a bug because it does not make sense showing the default parameters if they are not going to be used, especially when the short queries option is checked by default. Ideally, they should have not checked the short queries option and if it is checked by the user, the resulting default parameters should be shown.

So, if you don't plan to use the short queries option, just uncheck it and the parameters displayed will be used for the search; you can change them if you like. If you are happy with what you get on the results page, then take note of the parameters and then set them for use by the Network Blast.

Now, for those who plan to use the short queries option, do the following:

1. Don't bother modifying the default algorithm parameters and click on the submit button.
2. On the results page, click on the "Edit and Resubmit" button to find out what algorithm parameters were used for the search. The parameters that you see on the resulting page, were the exact values that were used for the search. If you are happy with the blast result, you can stick with these parameters, otherwise, you are free to modify them. If you plan to modify them, make sure you uncheck the option for short queries in order for your changes to be effective, otherwise, the default parameters for short queries that were displayed on that page will still be used. Once you are satisfied with your final set of parameters, take note of them and then set them for use by the Network Blast.

== Where to get Network Blast ==

ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/2.2.17/
File: netblast-2.2.17-ia32-freebsd.tar.gz 2334 KB 8/26/2007 4:09:00 PM
File: netblast-2.2.17-ia32-linux.tar.gz 2758 KB 8/26/2007 4:13:00 PM
File: netblast-2.2.17-ia32-solaris.tar.gz 2416 KB 8/27/2007 8:10:00 PM
File: netblast-2.2.17-ia32-solaris9.tar.gz 2455 KB 8/26/2007 4:15:00 PM
File: netblast-2.2.17-ia32-win32.exe 1479 KB 8/22/2007 9:40:00 PM
File: netblast-2.2.17-ia64-linux.tar.gz 5319 KB 11/1/2007 12:23:00 PM
File: netblast-2.2.17-mips64-irix.tar.gz 4340 KB 8/26/2007 4:17:00 PM
File: netblast-2.2.17-sparc64-solaris.tar.gz 4448 KB 8/26/2007 4:41:00 PM
File: netblast-2.2.17-universal-macosx.tar.gz 4398 KB 8/26/2007 4:25:00 PM
File: netblast-2.2.17-x64-linux.tar.gz 2834 KB 8/26/2007 4:13:00 PM
File: netblast-2.2.17-x64-solaris.tar.gz 3287 KB 8/27/2007 8:27:00 PM
File: netblast-2.2.17-x64-win64.exe 1686 KB 8/19/2007 4:29:00 AM

For other methods, see http://www.ncbi.nlm.nih.gov/BLAST/Doc/urlapi.pdf
and http://www.ncbi.nlm.nih.gov/blast/blast_overview.shtml
and the query.fcgi pages on how to construct an api to talk to various NCBI queries, e.g. automating the retrieval of GI numbers in FASTA or whatever formats.

== How to run Network Blast in Linux OS (e.g BioSlax)==

tar -zxvf netblast-2.2.17-ia32-linux.tar.gz

* The following will be untarred (unzipped): VERSION bin/ data/ doc/
* Blastcl3 is found in the bin folder

== Example of how to run Network Blast ==

1. Split the input fasta file (containing multiple sequences) into multiple files, with one sequence per file, using the command csplit. For example:

csplit -n 1 multiple.fasta /\>/ \{\*\}

The above command adds digits to names of the output fasta files without the padding of zeros infront.

* Example of content of the input fasta file:
>1
ESWILRNPGYALVA
>2
GCGLFGKGSIDTCA

..etc..

* Examples of output files:

file name: xx1
content of xx1:
>1
ESWILRNPGYALVA

file name: xx2
content of xx2:
>2
GCGLFGKGSIDTCA

.
.
.

file name: xx10
content of xx10:
>10
GCGLFGKGSIDTCA

and so on to the last sequence ...

2. Create an executable file containing the following code (relevant for queries of short length):

for i in xx*
do
blastcl3 -p blastp -d nr -i $i -F F -e 200000 -I T -v 0 -b 20000
-f 11 -W 2 -T F -u "All[FILTER] NOT Viruses[ORGN] NOT
txid81077[ORGN]" -C F -M PAM30 -o $i.out
sleep 10
done

'''NOTE:'''
* xx refers to the name of the input files, for example xx1, xx2, etc
* Sleep 10 is the time gap (10 seconds) between the end of one submission and the start of the next. This can be changed to longer time periods (recommended) in order not to overload the NCBI server. Otherwise, they may black list your IP.
* -p is blast p program, -d nr is the NR database, -i is for input filename, -F F for false is to switch off low complexity and other filters, -I T set to true is to show the GI numbers in the definition lines, and -v 0 is to set number of database sequences to show one-line descriptions, -b is to list out 20000 hits, -f 11 threshold for extending hits, -W 2 set word size for short sequence queries, default used if set to zero (blastn 11, megablast 28, all others 3, and 2 for short queries), -T F to produce text output, instead of HTML, -u "Root[ORGN] NOT txid11082[Organism:exp] NOT txid81077[ORGN]" is to restrict search of database to results of Entrez2 lookup [String], -C F to not use composition-based statistics for blastp or tblastn (0 or F or f: no composition-based statistics), -M PAM30 select matrix for short sequence queries (BLOSUM62 for normal protein searches), and -o to specify output filename.

3. Change the mode of the executable file in order to be able to execute. For example:

chmod 755 x

* x refers to the name of the executable file.
* For more information, see [http://en.wikipedia.org/wiki/Chmod Chmod]and [http://www.xaviermedia.com/documents/chmod755.php Chmod755].

4. Run the executable file. For example:

./x

* x refers to the name of the executable file

A task can usually be started and run as a background task by putting a '&' at the end of the command line. For example:

./x &

If a task was started and is running in the foreground, it is still possible to move it to the background without cancelling it. To move a task from the foreground to the background perform the following steps:

1. CTRL-Z (That is, while holding the CTRL key down, tap the 'z' key) This will suspend the current foreground job (task).
2. Enter the job control command 'bg'
3. Tap the 'Enter' key

The job is now running in the background.

Useful commands to see which jobs are still running is the 'jobs' or the 'ps ua' commands. If the 'jobs' command is used, a background jobs can be brought to the foreground with the command fg n where n is the job (not the PID) number.

==Manual pages of Network Blast==

blastcl3 2.2.17   arguments:

-p  Program Name [String]
-d  Database [String]
default = nr
-i  Query File [File In]
default = stdin
-e  Expectation value (E) [Real]
default = 10.0
-m  alignment view options:
0 = pairwise,
1 = query-anchored showing identities,
2 = query-anchored no identities,
3 = flat query-anchored, show identities,
4 = flat query-anchored, no identities,
5 = query-anchored no identities and blunt ends,
6 = flat query-anchored, no identities and blunt ends,
7 = XML Blast output,
8 = tabular,
9 tabular with comment lines
10 ASN, text
11 ASN, binary [Integer]
default = 0
range from 0 to 11
-o  BLAST report Output File [File Out]  Optional
default = stdout
-F  Filter query sequence (DUST with blastn, SEG with others) [String]
default = T
-G  Cost to open a gap (-1 invokes default behavior) [Integer]
default = -1
-E  Cost to extend a gap (-1 invokes default behavior) [Integer]
default = -1
-X  X dropoff value for gapped alignment (in bits) (zero invokes default behavior)
blastn 30, megablast 20, tblastx 0, all others 15 [Integer]
default = 0
-I  Show GI's in deflines [T/F]
default = F
-q  Penalty for a nucleotide mismatch (blastn only) [Integer]
default = -3
-r  Reward for a nucleotide match (blastn only) [Integer]
default = 1
-v  Number of database sequences to show one-line descriptions for (V) [Integer]
default = 500
-b  Number of database sequence to show alignments for (B) [Integer]
default = 250
-f  Threshold for extending hits, default if zero
blastp 11, blastn 0, blastx 12, tblastn 13
tblastx 13, megablast 0 [Real]
default = 0
-g  Perform gapped alignment (not available with tblastx) [T/F]
default = T
-Q  Query Genetic code to use [Integer]
default = 1
-D  DB Genetic code (for tblast[nx] only) [Integer]
default = 1
-a  Number of processors to use [Integer]
default = 1
-O  SeqAlign file [File Out]  Optional
-J  Believe the query defline [T/F]
default = F
-M  Matrix [String]
default = BLOSUM62
-W  Word size, default if zero (blastn 11, megablast 28, all others 3) [Integer]
default = 0
-z  Effective length of the database (use zero for the real size) [Real]
default = 0
-K  Number of best hits from a region to keep (off by default, if used a value of 100 is recommended) [Integer]
default = 0
-P  0 for multiple hit, 1 for single hit (does not apply to blastn) [Integer]
default = 0
-Y  Effective length of the search space (use zero for the real size) [Real]
default = 0
-S  Query strands to search against database (for blast[nx], and tblastx)
3 is both, 1 is top, 2 is bottom [Integer]
default = 3
-T  Produce HTML output [T/F]
default = F
-u  Restrict search of database to results of Entrez2 lookup [String]  Optional
-U  Use lower case filtering of FASTA sequence [T/F]  Optional
-y  X dropoff value for ungapped extensions in bits (0.0 invokes default behavior)
blastn 20, megablast 10, all others 7 [Real]
default = 0.0
-Z  X dropoff value for final gapped alignment in bits (0.0 invokes default behavior)
blastn/megablast 50, tblastx 0, all others 25 [Integer]
default = 0
-R  RPS Blast search [T/F]
default = F
-n  MegaBlast search [T/F]
default = F
-L  Location on query sequence [String]  Optional
-A  Multiple Hits window size, default if zero (blastn/megablast 0, all others 40 [Integer]
default = 0
-w  Frame shift penalty (OOF algorithm for blastx) [Integer]
default = 0
-t  Length of the largest intron allowed in a translated nucleotide sequence when linking multiple distinct alignments. (0 invokes default behavior; a negative value disables linking.) [Integer]
default = 0
-C  Use composition-based statistics for blastp or tblastn:
As first character:
D or d: default (equivalent to T)
0 or F or f: no composition-based statistics
1 or T or t: Composition-based statistics as in NAR 29:2994-3005, 2001
2: Composition-based score adjustment as in Bioinformatics 21:902-911,
2005, conditioned on sequence properties
3: Composition-based score adjustment as in Bioinformatics 21:902-911,
2005, unconditionally
For programs other than tblastn, must either be absent or be D, F or 0.
As second character, if first character is equivalent to 1, 2, or 3:
U or u: unified p-value combining alignment p-value and compositional p-value in round 1 only
[String]
default = D
-s  Compute locally optimal Smith-Waterman alignments (This option is only
available for gapped tblastn.) [T/F]
default = F

Tutorials to be posted

2009-04-01T23:57:00.000+08:00

This page was created to document the bioinformatics tutorial that we use and/or teach. Hopefully, by having all these tutorials documented, we will have a reference point in case there is a memory leakage, and also we can save time from teaching the same thing again and again by directing our students to this page first.

Below is a wish list of tutorials that I hope to document:

1. How to remove sequence duplicates from Fasta files
2. How to automate blast search using Netblast
3. How to use artemis
4. How to get representative sequences using blastclust
5. How to run phylogenetic tree using Phylip
6. How to run muscle alignment
7. How to use LibSVM
8. How to use Mega to identify conserved sequences considering including alignment positions with gaps and invariant residues
9. How to use client based blast
10. How to setup web blast using Bioslax
11. How to setup BioSlax as a webserver
12. How to configure MySQL in BioSlax
13. How to make backup of bioslax live CD
14. how to boot bioslax using thumb drive
15. How to make fasta file description lines unique for data downloaded directly from NCBI
16. How to use Meme
17. How to use HMM and create HMM profiles
18. How to use ANN
19. MS Excel shortcut/functions tutorials
20. MS Word shortcut/functions tutorials
21. How to configure network in Bioslax when it does not auto detect\
22. How to use PyMol to analyse exposed versus buried residues
23. How to use Swiss Model to model 3D structure
24. How to use Modeller to model 3D structure
25. How to use Rasmol
26. Blast parameters for short sequences
27. How to use TEPITOPE
28. Computing entropy using AVANA
29. Computing mutual information between two datasets using AVANA

anymore???....please add on and hopefully we will have post for each one of them in the near future!