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

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(PSIBLAST)
(PSIBLAST)
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** results mainly Aspartoacylases
 
** results mainly Aspartoacylases
   
 
*5 iterations, default Evalue 0.002
 
**<code> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it5_p45381_wt_big80.out -j 2</code>
 
** time ~5m
 
** 197 results
 
** Best E-Value E^-164
 
** Worst E-Value 7.9
 
** More significant results mostly Aspartoacylases, with higher E-Value (> 3E^-5) more Succinylglutamate Desuccinylases
 
   
   

Revision as of 22:05, 6 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

BLASTP

First, 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

We got 195 resulting sequences, out of which the last 101 have EValues below E^-15. 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.

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 -o hhblits_p45381_n10.out

-n number of iterations (def 2)
-e E Value CutOff (0.001)