1. What is Blast What/Why Standalone Blast Locating/Downloading Blast Using Blast You need: Your sequence to Blast and the database to search against

2. National Center for Biotechnology Information (NCBI) – 1988 BLAST – Basic Local Alignment Search Tool Blast finds regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases NCBI maintains several large annotated databases that are freely available to the public for download. YOU MAY WANT TO USE YOUR OWN DATABASE

3. Similarity search programs The BLAST Family blastp protein/protein blastn nucleotide/nucleotide blastx nucleotide/protein tblastn protein/nucleotide tblastx nucleotide/ nucleotide

4. http://www.ncbi.nlm.nih.gov/ Why Standalone Blast Why Standalone Blast Get Blast Here  Get Blast Here  Scroll Down To Here  Scroll Down To Here  Click FTP site Click FTP site Create a folder called Blast on your C:/ drive to store the downloaded file Create a folder called Blast on your C:/ drive to store the downloaded file The main advantage of Standalone Blast is to be able to create and Blast your own databases

5. Click Here  Click Here  Click Executables Click Executables Both Mac & Win

6. Click Latest  Click Latest  Choose 32-win32..  Choose 32-win32..  Or Mac users choose 32-linux.tar… 32-linux.tar… Click Save, then save the file in the Blast folder you created earlier Click Save, then save the file in the Blast folder you created earlier MAC Win Both

7. Go to the Blast directory: cd c:/ then cd /Blast Windows Install:./blast-2.2.20-ia32.win32.exe Go to the Blast directory: cd c:/ then cd /Blast Windows Install:./blast-2.2.20-ia32.win32.exe MAC Install: tar zxf blast-2.2.20-ia32-linux.tar.gz (MAC: you may find that an additional folder was created “blast-2.2.20”. In there you will find the folders below) MAC Install: tar zxf blast-2.2.20-ia32-linux.tar.gz (MAC: you may find that an additional folder was created “blast-2.2.20”. In there you will find the folders below) In the Blast directory there will now be 3 new folders In the Blast directory there will now be 3 new folders All Blast files are located here Blast documentation in html format. Best viewed in a web browser Algorithms for statistical analysis and sample search databases

8. Create the configuration files Create the configuration files In some instances, it may be necessary to restart your computer Go to your Windows directory (Win) cat > ncbi.ini [NCBI]Data="C:\Blast\data\“ TO SAVE & QUIT PRESS CTRL D Go to your root directory (Mac) cat >.ncbirc [NCBI]Data=/Blast/data SAVE & QUIT PRESS CTRL Z NOTE: This step appears to be unnecessary, and even problematic for newer versions of Windows and MAC OS X In some instances, it may be necessary to restart your computer

9. NOW YOUR BLAST PROGRAM HAS BEEN INSTALLED NOW YOUR BLAST PROGRAM HAS BEEN INSTALLED Test Blast using the single sequence and database provided ** Change to the BioDownload directory where your database and query sequence is stored. Formatdb Formatdb Prepare the database before Blasting To executeformatdb -i myDbase -p F you need the correct path -i the file to create into a searchable database -p T = protein database and F = nucleotide database /cygdrive/c/Blast/bin/formatdb -i./TA496Seq1.txt –p F

10. Formatdb creates index files Formatdb creates index files.nhr.nin.nsq Output Files with these extensions Blast Blast blastall -p blastn -d myDbase -i test.seq -o test.out blastall: access all blast programs blastn: select the blastn program since query and database are nucleotide -d: the database to blast -i: the query sequence(s) to blast -o: the name of the file to store your blast results /cygdrive/c/Blast/bin/blastall -p blastn -d./TA496Seq1.txt -i./TomatoSequence.seq –o out_tomato_blast

11. Additional Blast Parameters Additional Blast Parameters e-valuee Additional Blast Documentation Additional Blast Documentation Output of Blast Files & Parsing Output of Blast Files & Parsing