Difference between revisions of "Lab Journal - Task 3 (PAH)"

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== Secondary structure ==
  
== Secondary structure ==
  +
For P10775 three calls were done using ReProf. For the first one a FASTA file is used as input, whereas PSSM matrices are delivered for the other two. One created with PSI-Blast against the big80 database the other against swissprot. PSI-Blast is used with the same parameter like in Task2 with two iterations and an e-value cutoff of 10e-10(for big80: <code>blastpgp -i /mnt/home/student/waldraffs/Masterpraktikum/Task3/secondary_structure/<UniprotID>.fasta -d /mnt/project/rost_db/data/big/big_80 -j 2 -h 10e-10 -b 2000 -v 2000 -o check_out_files/<UniprotID>.out -Q swiss_matrix_<UniprotID>.pssm </code>, for swissprot only the database is changed: <code>-d /mnt/project/pracstrucfunc13/data/swissprot/uniprot_sprot)</code>.
  +
*<code> reprof -i <query>.fasta </code>
  +
*<code> reprof -i <query>.pssm </code>
  +
  +
Scripts created for this task:
  +
#[https://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php/Phenylketonuria/Task3/Scripts#filter_secStruc.pl filter_secStruc.pl]
  +
#[https://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php/Phenylketonuria/Task3/Scripts#SecStrucComparison.jar SecStrucComparison.jar]
  +
 
== Disorder ==
  
== Disorder ==
  +
  +
=== IUPred ===
  +
Before using this tool for the prediction, we had to compile IUPred with following command:
  +
cc /opt/iupred/iupred.c -o ''/mnt/home/student/.../iupred''
  +
  +
Afterwards one can invoke the programm as shown here:
  +
iupred ''sequence.fasta'' long|short|glob > ''output.txt''
  +
  +
Since the output is only given to Standard Out, we had to save the output into a file.
  +
  +
=== MD (MetaDisorder) ===
  +
To invoke the programm one can use following command:
  +
predictprotein --seqfile ''sequence.fasta'' --target metadisorder -p ''output_name'' -o ''output-directory''
  +
 
== Transmembrane helices ==
  
== Transmembrane helices ==
  +
  +
=== PolyPhobius ===
  +
Before using PolyPhobius, we had to do some steps:
  +
<ol>
  +
<li>We generated a fasta file with all homologous sequences to the query sequence inside. Therefore, we used the blastget perl script and the swissprot database as followed: <br /><pre> /mnt/project/pracstrucfunc13/polyphobius/blastget -db /mnt/project/pracstrucfunc13/data/swissprot/uniprot_sprot
  +
-ix /mnt/project/pracstrucfunc13/data/index_pp/uniprot_sprot.idx sequence.fasta > sequence-blast.fasta </pre>
  +
</li>
  +
<li>
  +
Afterwards, we used kalign for the MSA generation as shown here:
  +
<br /><pre> /mnt/opt/T-Coffee/bin/kalign -i sequence-blast.fasta -o sequence-kalign.fasta -f fasta
  +
</pre>
  +
</li>
  +
<li>
  +
Now, we can run PolyPhobius with following command:
  +
<br /><pre> /mnt/project/pracstrucfunc13/polyphobius/jphobius -poly sequence-kalign.fasta > sequence-polyphobius.txt
  +
</pre>
  +
</li>
  +
</ol>
  +
 
== Signal peptides ==
  
== Signal peptides ==
 
We tried two different parameters for our predictions:<br>
  
We tried two different parameters for our predictions:<br>
 
First we simple run SignalP without any constraints. The only thing, which has to be stated is ''-t euk'' as all four sequences are eukaryotic. Otherwise SignalP only would accept Gran+ or Gran-.
  
First we simple run SignalP without any constraints. The only thing, which has to be stated is ''-t euk'' as all four sequences are eukaryotic. Otherwise SignalP only would accept Gran+ or Gran-.
−
-o can be set, so the output is written automatically in output.txt or it can be set with '>'.
 +
-o can be set, so the output is written automatically in output.txt or it can be set with '>'.<br>
−
<code>signalp -t euk <UniprotID>.fasta > <UniprotID>_output.out </code>
 +
*<code>signalp -t euk <UniprotID>.fasta > <UniprotID>_output.out </code><br>
−
In our second run we choose only the N-terminal with 70 residues as it is recommended in the manual page of SignalP to avoid false positives.
+
In our second run we choose only the N-terminal with 70 residues as it is recommended in the manual page of SignalP to avoid false positives. <br>
−
<code>signalp -trunc 70 -t euk <UniprotID>.fasta > <UniprotID>_trunc.out</code>
 +
*<code>signalp -trunc 70 -t euk <UniprotID>.fasta > <UniprotID>_trunc.out</code><br>
−
In our case there are only few differences between the runs for the whole sequence or only the N-terminal. For example for the whole sequence the NN result of P47863 gives also a YES for C and not only for max.S. <xr id="signalp"/> shows the results of the N-terminal run only.
 +
In our case there are only few differences between the runs for the whole sequence or only the N-terminal. For example for the whole sequence the NN result of P47863 gives also a YES for C and not only for max.S. [[Sequence-based predictions (Phenylketonuria) #Signal peptides|Table 15]] shows the results of the N-terminal run only.
  +
  +
[[Category: Phenylketonuria 2013]]

Latest revision as of 13:41, 17 August 2013

Secondary structure

For P10775 three calls were done using ReProf. For the first one a FASTA file is used as input, whereas PSSM matrices are delivered for the other two. One created with PSI-Blast against the big80 database the other against swissprot. PSI-Blast is used with the same parameter like in Task2 with two iterations and an e-value cutoff of 10e-10(for big80: blastpgp -i /mnt/home/student/waldraffs/Masterpraktikum/Task3/secondary_structure/<UniprotID>.fasta -d /mnt/project/rost_db/data/big/big_80 -j 2 -h 10e-10 -b 2000 -v 2000 -o check_out_files/<UniprotID>.out -Q swiss_matrix_<UniprotID>.pssm , for swissprot only the database is changed: -d /mnt/project/pracstrucfunc13/data/swissprot/uniprot_sprot).

  • reprof -i <query>.fasta
  • reprof -i <query>.pssm

Scripts created for this task:

  1. filter_secStruc.pl
  2. SecStrucComparison.jar

Disorder

IUPred

Before using this tool for the prediction, we had to compile IUPred with following command: 

cc /opt/iupred/iupred.c -o /mnt/home/student/.../iupred

Afterwards one can invoke the programm as shown here: 

iupred sequence.fasta long|short|glob > output.txt

Since the output is only given to Standard Out, we had to save the output into a file.

MD (MetaDisorder)

To invoke the programm one can use following command: 

predictprotein --seqfile sequence.fasta --target metadisorder -p output_name -o output-directory

Transmembrane helices

PolyPhobius

Before using PolyPhobius, we had to do some steps:

  1. We generated a fasta file with all homologous sequences to the query sequence inside. Therefore, we used the blastget perl script and the swissprot database as followed: 
     /mnt/project/pracstrucfunc13/polyphobius/blastget -db /mnt/project/pracstrucfunc13/data/swissprot/uniprot_sprot 
 -ix /mnt/project/pracstrucfunc13/data/index_pp/uniprot_sprot.idx sequence.fasta > sequence-blast.fasta
  2. Afterwards, we used kalign for the MSA generation as shown here: 
     /mnt/opt/T-Coffee/bin/kalign -i sequence-blast.fasta -o sequence-kalign.fasta -f fasta
  3. Now, we can run PolyPhobius with following command: 
     /mnt/project/pracstrucfunc13/polyphobius/jphobius -poly sequence-kalign.fasta > sequence-polyphobius.txt

Signal peptides

We tried two different parameters for our predictions:
First we simple run SignalP without any constraints. The only thing, which has to be stated is -t euk as all four sequences are eukaryotic. Otherwise SignalP only would accept Gran+ or Gran-. -o can be set, so the output is written automatically in output.txt or it can be set with '>'.

  • signalp -t euk <UniprotID>.fasta > <UniprotID>_output.out

In our second run we choose only the N-terminal with 70 residues as it is recommended in the manual page of SignalP to avoid false positives.

  • signalp -trunc 70 -t euk <UniprotID>.fasta > <UniprotID>_trunc.out

In our case there are only few differences between the runs for the whole sequence or only the N-terminal. For example for the whole sequence the NN result of P47863 gives also a YES for C and not only for max.S. Table 15 shows the results of the N-terminal run only.

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