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

From Bioinformatikpedia
(Summary and Comparison)
(HHBlits)
Line 127: Line 127:
 
Regarding the Sequence Identity distribution, running HHBlits with 8 iterations results in more distant related Hits (see Figure ??).
 
Regarding the Sequence Identity distribution, running HHBlits with 8 iterations results in more distant related Hits (see Figure ??).
   
  +
[[File:CD_HHBlits_seqid_distri.png|thumb|center|blabla]]
[[File:Example.jpg]]
 
   
 
===Comparison of found sequences ===
 
===Comparison of found sequences ===

Revision as of 16:51, 7 May 2012

Task 2 : Canavans Disease

Sorry, guys, we're a bit behind schedule! Hope to have everything finished before 11pm tonight (Monday) and hope that's early enough for you to read. Sorry again! Susi and Fanny

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 of the runs with 10 iterations have lower E-Values than the best hits of the runs with less iterations. Yet, the result includes a larger amount of significant hits with higher E-Values. This means, increasing the iterations finds further distantly related sequences, which is the expected outcome. This outcome is also represented in the distribution of sequence identities. As one can see in figure ??, running PSI-Blast with 10 iterations results in hits with a lower sequence identity to our query sequence than the hits from the run with 2 iterations.

Figure ??
Distribution of Sequence Id between Psi-Blast runs with 2 iterations vs 10 iterations (using E-Value 10e-10)

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.

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
Figure ??
Distribution of Sequence Id for Psi-Blast runs with 2 iterations with different E-Values (def E-Value vs E-Value of10e-10)
Figure ??
Distribution of Sequence Id for Psi-Blast runs with 10 iterations with different E-Values (def E-Value vs E-Value of10e-10)

HHBlits

Running HHBlits with 2 iterations yields a small amount of hits (270) with very low (2e-110) and very high (0.0011) E-Values. To increase the amount of hits, we repeted the HHBlits search with the maximum amount of 8 iterations which resulted in a broader output with more Hits with lower averaged E-Values. Regarding the Sequence Identity distribution, running HHBlits with 8 iterations results in more distant related Hits (see Figure ??).

blabla

Comparison of found sequences

Default E-Values: as could be expected, the normal BLAST search is mostly contained in the PsiBLAST search with two iterations. HHBlits found a large number of different hits, with only 48 out of 274 common hits in common with the BLAST searches.
Taking PsiBLAST with 10 iterations into account brings in a large number of common sequences among the three searches (110), which could be interesting since there seems to be high conversation among them.
Strict E-Values for PsiBLAST and default E-Value for HHBlits with 2 iterations: The number of common hits among all three is now substantially lower, while PsiBLAST with two and ten iterations share a great number of their hits.
Increasing the number of HHBlits-iteration yields more hits for HHBlits, but does not increase the number of common hits with PSI-Blast in 2 or 10 iterations. However, 10 sequences are common and could be interesting for further investigation.

}