PHYLIP is a free package of programs for inferring phylogenies. It is distributed as source code, documentation files, and a number of different types of executables. These Web pages, by Joe Felsenstein of the Department of Genome Sciences and the Department of Biology at the University of Washington, contain information on PHYLIP and ways to transfer the executables, source code and documentation to your computer.

Let's assume that you have fetched the appropriate executables archives (if any) for your machine, together with the Documentation and Sources archive.

These archives are not simply a single executable for general use. Instead they are a (large) number of files, squished together into one file, and thus called an "archive". You have to go through a step which involves getting all the files out of the archive and into a folder on your machine.

We will give these instructions for four different kinds of systems, Windows, Mac OS X, Linux or Unix systems, and Mac OS 8 or 9. Then we will describe how to access the documentation after installing the package.

Windows
Our main method of distributing PHYLIP for Windows systems is in a self-extracting archive. A self-extracting archive has a small self-extraction program on its front, and when you run them as programs they extract themselves and write many files into your folder.

On the Windows system you will have a self-extracting archive called phylip-3.69.exe. Place it in a folder where you will want PHYLIP to reside. After it self-extracts, it will make a folder called phylip-3.69 there, in the folder where you place the self-extracting archive. So if you place the archive on the Desktop, it will make that folder there.

To self-extract the archive simply double-click on it. If your Windows system does not allow the archive to self-extract, you may have to right-click on the archive and select "Run as administrator". Accept the default settings, making sure that it will extract into the folder that you want it in, and when a dialog box comes up, click on Unzip. When the extraction is complete, select Close. You will find the folder phylip-3.69 in the same place as the archive.

After the self-extraction is done, you will find the phylip-3.69 folder which has three folders within it, called doc, exe, and src. There is also a web page phylip.html. You should read this in your browser. It contains links to the main documentation web page main.html and to the other documentation pages and programs.

Note: There has been a report that on Windows Vista (specifically the report was for Service Pack 2 on Home Edition) that if you extract the files into a folder that is somewhere under Program Files, they may not work properly. You can avoid this problem if you extract them into a folder which is somewhere in your own folders.

Macintosh Mac OS X

The archives for Intel iMac and PowerMac Mac OS X are in the Apple .dmg disk image format. There is one .dmg disk image distributed. It is 6.8 Megabytes in size, and is compressed; when uncompressed it is several times larger. (Note that Mac OS X version 10.0 did not support compressed disk image files, so the .dmg file we distribute will not work on it).

It contains what looks like a normal folder called phylip-3.69, but you should not use the programs while they are in that folder. It is important that you use a copy of the contents of the disk image elsewhere on your system. Decide where the folder phylip-3.69 is going to reside, and copy that folder to that new location. Within it is all the source code, documentation, and executables.

The executables are 32-bit "universal binaries" that will run on either the newer iMacs (which has Intel processors) or the older G3's, G4's or G5's that have PowerMac processors. (Basically they are each simply a pair of executables set up so that the processor chooses the right one and runs it).

There is also a set of links in the exe folder to enable you to run the programs by simply typing their name. If these are absent, which they should not be, there is one additional set of steps you need to do:
Open a Terminal window (which you will find in the Utilities folder of the Applications folder).
At the prompt, change to the folder where the PHYLIP executables are kept. For example, if you have folder phylip-3.69 on your desktop, type in the command
cd Desktop/phylip-3.69/exe
Change the permissions of the script linkmac which you will find there to allow it to execute:
chmod +x linkmac
Then execute the script by typing: ./linkmac

The result will be that the executables folder will now have a set of links, one for each program, that allow the programs to be executed simply by typing their names. Thus the programs in that folder can then be executed either by clicking on their icon, or by typing their name (in lower case) preceded by ./ to indicate the current folder, such as ./dnaml

This setting up of these links needs to done only once, and they will be available thereafter.

Linux and Unix

You can easily install PHYLIP and compile it yourself on a Linux or Unix system, provided that you have a C compiler on your system. (On some Linux systems and in some recent versions of Mac OS X the C compiler is not installed by default, and you may have to make sure that it is installed).

The archive for Linux or Unix systems is just the Documentation and Sources archive. You can extract the files from the archive by putting it into a folder by itself, getting into that folder, and giving the command

gunzip phylip-3.69.tar.gz

This uncompresses the archive so that it will now be bigger and be called phylip-3.69.tar. Now it is a "tar archive".

Now just issue the command

tar xvf phylip-3.69.tar

The archive will be extracted and a phylip3.69 folder created that contains within it three folders, doc, exe, and src, as well as a web page phylip.html. You should start by reading that web page. It contains links to the documentation web pages, including the main documentation web page main.html.

To make executables, use your C compiler. It is probably as simple as going into the src directory and typing the command

make install

which will cause the programs to be compiled and the executables and font files installed in the exe directory. If there are problems with the compilation, read the relevant sections of main.html and also the beginning of the Makefile.

Compiling on Ubuntu (or other Debian-family) Linux

I have found that to compile PHYLIP 3.69 on an Ubuntu system, and presumably on other Debian-family Linux releases, you need to load some X11 libraries before compiling. Try the following:
Make sure you have enough administrative privileges for the following (or go get the help of those that do).
Load the X11, Xt and Xaw libraries by doing these commands:

3. sudo apt-get install libX11-dev

4. sudo apt-get install libxt-dev

5. sudo apt-get install libxaw7-dev
If all those installations work, you can then get into the src folder, and as indicated above, just execute the command

7. make install

With luck this will work. After the compilation the executables and their font files will be in folder exe.

Macintosh Mac OS 8 or Mac OS 9

We don't think there are many Mac OS 8 or Mac OS 9 machines around any more -- Apple has abandoned that operating system, and it is probably not wise to use it on the Internet as it has not been upgraded to cope with recent security threats. We no longer distribute Mac OS 8 or 9 executables for our most recent versions of PHYLIP. Nevertheless, we do make available a Stuffit (.sit) archive of executables for PHYLIP 3.65, just in case anyone needs it.

Downloading the single Stuffit archive

The archives for Mac OS 8, Mac OS 9 Macintoshes are self-extracting archives. If you are fetching the files using a Web browser directly from your Mac, it will probably automatically invoke Stuffit Expander to unpack the archives into self-extracting archives. Self-extracting archives are archives with a small self-extraction program on their front, so that when they are run as programs they write a great many files into a folder.

Our default distribution is of a single large (4.9 Megabyte) Stuffit archive called phylip3.65.sit. This should automatically be unstuffed as it is downloaded. It contains the source code, documentation, and Mac OS executables.

However, at the moment we have reports of trouble running the unstuffed archives. Recent versions of Mac OS 9, and also the Classic environment under Mac OS X, seem to regard the executables files as text files, and they try to open them under SimpleText, which of course does not work.

Downloading multiple smaller Stuffit archives

Some people may wish instead to download these as a series of four archives. This might be necessary, for example, if you wanted to transfer them to a machine that was not connected to the Internet, and needed them each to be small enough to write onto a floppy disk.

If you choose to download these instead of the single Stuffit archive, and our system does automatically invoke Stuffit Expander, four self extracting archives will be created on your desktop, and their file names will end in .sit. If you have Stuffit Expander, you can use it to expand them.

Once the archives are downloaded and extracted, they will put folders on the desktop. One is called phylip and contains folders doc and src as well as a web page phylip.html. There are 307 files in all, as these include the documentation files, source code files, and compilation support files. You may have to be patient while the archive is being extracted.

The three archives of Macintosh Mac OS 8 and Mac OS 9 executables will create folders exe, exe.1, exe.2 and exe.3 on the desktop when they are extracted. For some reason we could not create these inside the phylip folder, but that is where these files need to be put. Do the following:

1. Move the exe folder inside the phylip folder.
2. Open the exe.1 folder, and mark all of its files (you can do this using the Select All menu option in the system Edit menu).
3. Drag this group of marked files into the exe folder which is now inside the phylip folder.
4. Now do the same for exe.2 (mark the files and drag them into exe).
5. And do the same for exe.3 (mark the files and drag them into exe).
6. You can now discard the folders exe.1, exe.2 and exe.3 (if any). If you have four folders named phylip3.65.0.sit, exe.1.sit, exe.2.sit, and exe.3.sit on your desktop, these can be discarded too.
7. The phylip3.65 folder can now be moved to wherever you want it to be.

Source: http://evolution.gs.washington.edu/phylip

MEGA (Molecular Evolutionary Genetic Analysis)

MEGA is an integrated tool for conducting automatic and manual sequence alignment, inferring phylogenetic trees, mining web-based databases, estimating rates of molecular evolution, and testing evolutionary hypotheses.

MEGA 4/ MEGA 5 has been tested on the following Microsoft Windows® operating systems:
Windows 95/98, NT, 2000, XP, Vista, and Windows 7.

Necessary step:

1. Go to http://www2.uwrf.edu/caseit/tutorialv5/download2.html, and follow the steps to register the free Case It software. You will then go to the download page. Select Case It v6, and save the compressed file to your desktop. Then, double-click on the zipped file and extract it to your desktop. A folder called 'Case It v6' will appear as shown below (the Case It executable is inside this folder, called 'Case It v6.exe').

---

Optional steps: Installation of MEGA 4 to work in conjunction with Case It (necessary for tree-building).
2. Go to http://www.megasoftware.net/mega.html. Register the free MEGA 4 software by filling in your name and email address, select 'Download zip file only', and click the 'Download' button. The following message will appear. Save to your desktop, and the icon to the right will appear on your desktop.

---

3. Right-click on the desktop icon and select 'Extract All...'. Extract the application to your desktop. An icon called 'setup.exe' will appear.

---

4. Double-click on the 'setup.exe' icon, and install the installation package on your desktop.

---

5. When the screen below appears, click the 'Change' button and navigate to your desktop to find the folder



Double-click to open the Case It v6 folder...


...and locate the folder MEGA that is inside the Case It v6 folder. Double-click on the MEGA folder to open it...


...and click OK to install the MEGA 4 application and folders

---

6. To make sure that the MEGA 4 materials were installed correctly, go to your desktop and double-click on the Case It v6 folder to open it.

Double-click on the MEGA folder to open it...


...and you should see the MEGA4.exe application and 3 folders, as highlighted below. The other files were already present inside the MEGA folder of Case It - these files are necessary so that Case It will work directly with MEGA 4.

Primer 3

Primer3 is a free online tool to design and analyze primers for PCR and real time PCR experiments. Primer3 can also select single primers for sequencing reactions and can design oligonucelotide hybridization probes. The online tool constitutes some important features like primer detection, cloning, sequencing and Primer listing.

Link: http://frodo.wi.mit.edu/

NCBI has provided BLAST sequence analysis services for over a decade. For many users, the first question they often face is "Which BLAST program should I use?" In order to help users arrive at an answer to this question, we created this "BLAST Program Selection Guide."

This document first introduces the BLAST databases available from NCBI (in Section 2). The actual guide (Section 3) divides BLAST searches into several categories according to the nature and size of the input query and the primary goal of the search. Starting from the query sequence column on the left and cross-referencing to the right, a user will arrive at the specific BLAST program(s) best suited for that search.

This document is also available in PDF (163,516 bytes).

BLAST Database content

A BLAST search has four components: query, database, program, and search purpose/goal. To discuss effective BLAST program selection, we first need to know what databases are available and what sequences these databases contain. In this section, we will first take a look at the common BLAST databases. According to their content, they are grouped into nucleotide and protein databases.

MEGABLAST is the tool of choice to identify a nucleotide sequence

The best way to identify an unknown sequence is to see if that sequence already exists in a public database. If the database sequence is a well-characterized sequence, then one will have access to a wealth of biological information. MEGABLAST, discontiguous-megablast, and blastn all can be used to accomplish this goal. However, MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences and thus is the best tool to use to find the identical match to your query sequence. In addition to the expect value significance cut-off, MEGABLAST also provides an adjustable percent identity cut-off for the alignment, which provides cut-off in addition to the significance cut-off threshold set by Expect value.

Web MEGABLAST and discontiguous megablast pages can also accept batch queries, the only web BLAST pages with this capability. Please refer to the "Batch Search" section for details.

Discontinuous MEGABLAST is better at finding nucleotide sequences similar, but not identical, to yours nucleotide query 

The BLAST nucleotide algorithm finds similar sequences by breaking the query into short subsequences called words. The program identifies the exact matches to the query words first (word hits). BLAST program then extends these word hits in multiple steps to generate the final gapped alignments. 

One of the important parameters governing the sensitivity of BLAST searches is the length of the initial words, or word size as it is called. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). Because of this, blastn is better than MEGABLAST at finding alignments to related nucleotide sequences from other organisms. The word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity. 

A more sensitive search can be achieved by using the newly introduced discontiguous megablast page. This page uses an algorithm with the same name, which is similar to that reported by Ma et.al. Rather than requiring exact word matches as seeds for alignment extension, discontiguous megablast uses non-contiguous word within a longer window of template. In coding mode, the third base wobbling is taken into consideration by focusing on finding matches at the first and second codon positions while ignoring the mismatches in the third position. Searching in discontiguous MEGABLAST using the same word size is more sensitive and efficient than standard blastn using the same word size. For this reason, it is now the recommended tool for this type of search. Alternative non-coding patterns can also be specified if desired. Additional details on discontiguous are available at: 
www.ncbi.nlm.nih.gov/Web/Newsltr/FallWinter02/blastlab.html 

Useful links:

Protein BLAST

Nucleotide BLAST

Translated BLAST: blastx

ClustalW2 is a general purpose multiple sequence alignment program for DNA or proteins. 

To use this tool, click on this link.