Stand alone BLAST on Linux

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Stand alone BLAST on Linux
CBW Bioinformatics Vancouver 2004 Lab 4.1
Sohrab Shah bioinformatics.ubc.ca sohrab_at_bioinfo
rmatics.ubc.ca
Stephanie Minnema University of Calgary Will
Hsiao Simon Fraser University
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Outline

• What is stand alone BLAST?
• Why stand alone BLAST?
• Installing BLAST
• Formatting databases for BLAST
• Running stand alone BLAST searches
• Changing parameters
• Formatting BLAST output
• Assignment

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What is stand alone BLAST?

• A local installation of the NCBI BLAST suite of
  programs
• Requires CPU, disk and RAM
• The same application that drives the NCBI WWW
  BLAST server
• Software distribution and documentation available
  from
• ftp//ftp.ncbi.nih.gov/blast/executables/release

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Why stand alone BLAST?

• Allows creation of custom databases
• Specific data sets for specific tasks
• Increase computational efficiency
• Increase specificity of results
• Secure querying
• Important for IP protection no internet traffic
• Facilitates high-throughput analyses
• No queues only competing with internal users
• Can automate searches

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Some drawbacks

• Often need significant hardware resources
• Need to maintain the databases

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Installing BLAST

• The BLAST distribution
• Point your browser to
• ftp//ftp.ncbi.nih.gov/blast/executables/release
• Mailing list
• http//www.ncbi.nlm.nih.gov/mailman/listinfo/blast
  -announce
• Distribution announcements
• Bug reports/fixes

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ftp//ftp.ncbi.nih.gov/blast/executables/release/2
.2.6
We have already downloaded the distribution, but
this is the ftp directory
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Installing BLAST
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Unpack the distribution
Unzip the distribution
Helpful info Standalone BLAST is distributed as
a gziped tar archive The .gz file extension
indicates that the file has been compressed with
gzip a standard Unix compression utility The
gunzip utility uncompresses the file See gt man
gunzip for more info
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Unpack the distribution
Untar the distribution

• Helpful info
• The .tar extension indicates that the file is a
  tape archive created with tar a standard Unix
  archiving tool
• The tar command above extracts the archive into
  the current working directory
• See gt man tar for more info
• x extract
• p preserve permissions
• f file

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List the contents of the distribution

• A suite of tools for
• running various blast searches
• formatting and extracting sequences
• Documentation
• README. files read em!
• Data files with scoring matrices
• data

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Configuring BLAST

• We need to configure the system so the BLAST
  programs can function correctly
• Set the PATH environment variable by editing
  /.bashrc

Save the file
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Configuring BLAST

• We need to set up a configuration file /.ncbirc
  to point to the data directory in the
  distribution
• Open a file
• emacs /.ncbirc
• Save the file

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Exit the shell

• Exit the shell
• Start a new shell
• When you start a new shell, your environment will
  be set up to run BLAST

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Formatting the swissprot database for BLAST

• Change directory to /home/guest/blast/db
• View the contents of the directory
• Unzip the swissprot database

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View the contents of the swissprot database
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FASTA format
gtSOME DEFINITION OF THE SEQUENCE
\n ACGATCGACTACGATCAGCAGCATAGCTACAGATAG
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FASTA -gt BLASTable

• FASTA formatted files are not compatible for the
  BLAST programs
• You need to prepare the FASTA files for BLAST
  with formatdb
• This indexes the entries in the FASTA file and
  enables BLAST to run much faster

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formatdb

• Formats FASTA formatted databases for BLAST

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Formatting swissprot

• Format the swissprot database using formatdb
• List the contents of the directory
• The formatdb command will take a few minutes

• Useful info
• there should be seven files that are a
  combination of indexes and data
• note the formatdb.log file
• View its contents with more formatdb.log
• Ignore WARNING errors potential bug in new
  release
• You should see Formatted 143046 sequences in
  volume 0 as the last line in the file

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formatdb documentation
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Running BLAST - parameters
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Running BLAST - parameters
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Running BLAST - parameters
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Running BLAST - parameters
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Running BLAST try it

• Change directory to /home/guest/Lab4.1
• List the contents

• Useful info
• bact_genome.fna 12Kb of genomic sequence of
  Pseudomonas aeruginosa for the assignment
• hs_tryp_trna_synth.aa Human tryptophanyl tRNA
  synthetase to try command psi-blast
• test_blast.aa test protein to try blastp and
  rpsblast
• unknown1.aa mystery protein for assignment
• unknown2.aa mystery protein for assignment

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Running BLAST try it
Run the blastall command below What will this
command do?
What is the protein in test_blast.aa? Repeat the
search with a higher e-value cut-off (10) . How
does the output change?
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BLAST output
NEW
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BLAST output
NEW
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rpsblast

• Reverse Position Specific BLAST
• Query protein sequence
• Database domains
• We have installed Pfam on your laptop
• http//pfam.wustl.edu/
• Other domain databases Smart
• http//smart.embl-heidelberg.de/
• CDD
• http//www.ncbi.nih.gov/Structure/cdd/cdd.shtml
• For creating local blastable domain databases,
  consult
• ftp//ftp.ncbi.nih.gov/pub/mmdb/cdd/README

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Running rpsblast
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Run rpsblast

• Search test_blast.aa against Pfam
• Produce HTML output with T
• Open the results in your browser

What domains are present?
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rpsblast output
NEW
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Running psiblast

• Preferred option when dealing with an unknown
  protein
• or trying to find distant homologues
• Much more sensitive than blastall
• Less specific with each iteration
• Use blastpgp to run psiblast on the command line

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blastpgp parameters
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blastpgp parameters
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blastpgp parameters
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blastpgp parameters
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Running psiblast (blastpgp)

• Search swissprot with human tryp tRNA synthetase
  using psiblast with 4 iterations. Generate HTML
  output

How does the hit list change with each
iteration? How can the matrix.ctx file be used in
downstream analysis?
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psiblast results
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Further information

• Consult README files in BLAST distribution

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Summary

• A standalone BLAST server enables custom, secure,
  high throughput searches
• BLAST distribution available from
• ftp//ftp.ncbi.nih.gov/blast/executables/release
• Use command line parameters to tune your
  searches and format your results
• Use different BLAST tools for different purposes
• Regular (blastall blastp, blastn, blastx,
  tblastn, tblastx)
• Searching for domains (rpsblast cdd search)
• distant homologues (blastpgp psi/phi blast)

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Assignment

• Four questions
• Running
• blastp identify a protein
• rpsblast search for domains in a protein
• blastx annotate a genomic sequence
• psiblast find a function for an unknown protein
• Some searches may take a few minutes
• Where applicable report the e-value of hits and
  their locations on the query sequence and the
  command you used to run the search
• No longer than 2 printed pages
• Submit to Saara by Fri 9am