Difference between revisions of "Canavan Task 2 - Sequence alignments"

From Bioinformatikpedia
m (BLASTP)
(BLASTP)
Line 23: Line 23:
 
==== BLASTP ====
 
==== BLASTP ====
 
We ran BlastP on student machines with the big_80 as a reference database.
 
We ran BlastP on student machines with the big_80 as a reference database.
  +
  +
Command:
  +
<code>blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out</code>
  +
   
 
<table border=1 cellspacing = 0>
 
<table border=1 cellspacing = 0>
Line 32: Line 36:
   
 
</table>
 
</table>
 
Command:
 
 
<code>blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out</code>
 
 
*default E-Value cutoff (10)
 
**196 resulting sequences, out of which the last 101 have EValues below e-15.
 
**best E-Value: e-155
 
**worst E-Value: 9.6
 
**Most of the resulting proteins are Aspartoacylases of other species. Most of the results with EValue > e-15 are Succinylglutamate Desuccinylases, which catalyze a reaction similar to Aspartoacylase.
 
 
*more strict E-Value cutoff of 10e-10
 
**94 results
 
**best E-Value: e-155
 
**worst E-Value: e-15
 
**The results are the same as for the first run, just with an earlier cutoff
 
   
 
====PSIBLAST====
 
====PSIBLAST====

Revision as of 12:03, 7 May 2012

Task 2 : Canavans Disease

Sequence

The native ASPA sequence that we used for the current task is shown below:

UniProt: P45381

>hsa:443 ASPA, ACY2, ASP; aspartoacylase; K01437 aspartoacylase [EC:3.5.1.15] (A)
MTSCHIAEEHIQKVAIFGGTHGNELTGVFLVKHWLENGAEIQRTGLEVKPFITNPRAVKK
CTRYIDCDLNRIFDLENLGKKMSEDLPYEVRRAQEINHLFGPKDSEDSYDIIFDLHNTTS
NMGCTLILEDSRNNFLIQMFHYIKTSLAPLPCYVYLIEHPSLKYATTRSIAKYPVGIEVG
PQPQGVLRADILDQMRKMIKHALDFIHHFNEGKEFPPCAIEVYKIIEKVDYPRDENGEIA
AIIHPNLQDQDWKPLHPGDPMFLTLDGKTIPLGGDCTVYPVFVNEAAYYEKKEAFAKTTK
LTLNAKSIRCCLH



Search

BLASTP

We ran BlastP on student machines with the big_80 as a reference database.

Command: blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out


Parametersdefault E-Value = 10 E-Value 10e-10
results19694
best E-Value1e-1551e-155
worst E-Value9.6e-15
commentMost of the resulting proteins are Aspartoacylases of other species. Most of the results with EValue > e-15 are Succinylglutamate Desuccinylases, which catalyze a reaction similar to Aspartoacylase.The results are the same as for the first run, just with an earlier cutoff

PSIBLAST

PSIBlast was used in the same fashion as BLAST, with the big_80 as the background database. Results:

  • Running 2 iterations and default E-Value 0.002
    • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_p45381_wt_big80.out -j 2
    • time: ~2m30
    • 500 results
    • best E-Value: E^-142
    • worst E-Value: 3E^-4
    • Results with best EValues are mostly Aspartoacylases
    • Sequences previously not found are mostly Succinylglutamate Desuccinylases


  • 2 iterations, more strict E-value cutoff of 10E-10
    • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_h10e10_p45381_wt_big80.out -j 2 -h 10e-10
    • time ~2m30
    • 93 results
    • best E Value: E^-145
    • worst E Value: 2E-29
    • results mainly Aspartoacylases


  • 10 iterations, default Evalue 0.002
    • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_p45381_wt_big80.out -j 10
    • time: ~10m
    • converged after 8 rounds
    • 500 results
    • best E-Value 5E^-70
    • worst E-Value 8E^-38
    • most significant results include more Succinylglutamate Desuccinylases than Aspartoacylases


  • 10 iterations, E-value cutoff 10E-10
    • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_h10e10_p45381_wt_big80.out -j 10 -h 10e-10
    • time: ~10m
    • all 10 iterations were done (no early convergence)
    • 500 results
    • best E-Value: 7E^-70
    • worst E-Value: 1E^-38
    • aspartoacylases slightly more frequent in lower E-Values (< E-58), but no significant difference in E-Values for aspas and succis

HHBLITS

Run HHBlits on student machines with Uniprot20 database.

  • hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out
    • time: 2m50
    • 274 results
    • best E-Value: 2e-110
    • worst E-Value: 0.0011
    • mixed results with Aspa and Succi
  • hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -o hhblits_p45381_n10.out
    • time: 6m
    • 500 results
    • best E-Value: 2.9e-68
    • worst E-Value: 9.5e-09
    • very varying results: Aspartoacylasen, Succinylasen, Zinc Proteins

-n number of iterations (def 2)


Summary and Comparison

Along with the expactations one can find more hits with Psi-Blast than with a simple Blast search.

In general, one can distinguish between two kinds of proteins, that frequently are identified by the sequence searches:

  • Aspartoacylases
  • Succinylglutamate Desuccinylases

Increasing the amount of iterations performed in a PSI-Blast search, obviously increases the running time. One can see, that the best ranked hits have lower E-Values than the best Hits of the runs with less iterations. Yet, there are more hits found with better E-Values, which is not surprising because more homologues of significant profile sequences will be found.

When restricting the E-Value Cutoff for the profile built-up, we found that more hits are classified as Aspartoacylases than as Succinylglutamate Desuccinylases. The running time, as well as the E-Values of the resulting hits did not change significantly.

Comparison of found sequences

hello