Exercises

Search for homologies with hidden Markov models

Obtain the Swissprot-entry of the myb proto-oncogene protein (AC P10242, entry MYB_HUMAN).

Take the amino acid sequence of the myb protein and search against proteins with BLAST.

1. Concatenate domains

   We want to obtain a HMM for myb-domains to search against a database. Keep that in mind while screening the hits of the BLAST-search. Select some myb-domains and copy the corresponding parts of the sequences to a file in fasta-format (fasta format is described in exercises section of Pairwise Alignments).

   
   

2. Multiply align domains

   Download ClustalW (or ClustalX ?) from EBI-ftp-Server:
   -> ftp.ebi.ac.uk
   -> directory /pub/software/unix
   -> clustalw.tar.Z

   decompress and detar with
   
   $ gtar xvzf clustalw.tar.Z
   $ cd clustalw1.7

   compile the source code distribution:

   $ make

   Multiply align the myb-domains by clustalw.
   
   
   

3. HMMER

   Install HMMER 2.1.1

   Download from

   -> ftp.genetics.wustl.edu
   -> directory /pub/eddy/hmmer
   -> hmmer-2.1.1.tar.Z

   decompress, detar (see above) and install source code distribution (see file INSTALL):

   $ cd hmmer-2.1.1
   $ ./configure
   $ make

   The binaries now are in the directory binaries.

   
   

4. Build HMM of myb-domains

   Build and calibrate a HMM of the myb-domains by means of hmmbuild and hmmcalibrate.

   
   

5. Search a database for homologues
   

   We want to search a fasta-database consisting of concatenated sequences in fasta format (fasta format is described in exercises section of Pairwise Alignments).
   Therefore, first create a directory for the database and change into this directory:
   
   $ mkdir database
   $ cd database

   Download e.g. the peptides of the Drosophila genomes in fasta format from NCBI (~7,5 MB)

   -> ftp ncbi.nlm.nih.gov (ftp-Server at NCBI)
   -> directory /genbank/genomes/D_melanogaster/Scaffolds/LARGE
   -> download all files ending with '.faa'

   concatenate the peptides of all scaffolds into one file:

   $ cat *.faa > drosophila.fasta
   ($ rm *.faa)

   Use hmmsearch to search against a database with the HMM of the myb-domains:
   Use '>' to direct standard output to a file.
   The commandline may look like this:
   

   $ hmmsearch myb_domains.hmm ~/database/Drosophila.fasta > myb_hmm_drome.log
   
   
   

6. Iterate search

   Screen the hits, build a new HMM including selected hits and hmmsearch again.

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   Comments are very welcome.
   luz@molgen.mpg.de