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

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(BlastP)
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===BlastP===
 
===BlastP===
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[[File:CD_blast_seqid_distri.png|thumb|300px|right|Comparison of distribution of Sequence Identiy between the two BlastP runs ]]
 
 
A simple blast search yields only about 90 significant hits it one considers a threshold of 10e-10 as a significance cutoff.
 
A simple blast search yields only about 90 significant hits it one considers a threshold of 10e-10 as a significance cutoff.
  +
As one can see in Figure ??, the restriction of the Evalue results in less hits with a low sequence similarity.
[[File:CD_blast_seqid_distri.png|thumb|300px|center|Comparison of distribution of Sequence Identiy between the two BlastP runs ]]
 
   
 
===Psi Blast===
 
===Psi Blast===

Revision as of 12:36, 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. Commands:

  • 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


  • 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


  • 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


  • 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


Parameters it2, def E-Value (0.002) it2 E-Value 10e-10 it10 def E-Value (0.002)it10 E-Value 10e-10
time ~2m30 ~2m30 ~10m time: ~10m
results 500 93 500 500
best E-Value1e-142 1e-145 5e-70 7e-70
worst E-Value3e-4 2e-29 8e-38 1e-38
commentsResults with best EValues are mostly Aspartoacylases, Sequences previously not found are mostly Succinylglutamate Desuccinylasesresults mainly Aspartoacylases- converged after 8 rounds
- most significant results include more Succinylglutamate Desuccinylases than Aspartoacylases
- all 10 iterations were done (no early convergence)
- 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.


Commands

  • 2 iterations:
    • hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out
  • 8 iterations:
    • hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -o hhblits_p45381_n10.out

-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

BlastP

Comparison of distribution of Sequence Identiy between the two BlastP runs

A simple blast search yields only about 90 significant hits it one considers a threshold of 10e-10 as a significance cutoff. As one can see in Figure ??, the restriction of the Evalue results in less hits with a low sequence similarity.

Psi Blast

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.

HHBlits

Comparison of found sequences

Parametersit 2 it 8
time2m50~6m
results274500
best E-Value2e-1102.9e-68
worst E-Value0.00119.5e-09
commentmixed results with Aspartoacylases and Succivery varying results: Aspartoacylasen, Succinylasen, Zinc Proteins
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