Sequence Alignments HEXA

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Sequence Alignments

Sequence Searches

  • FASTA

/bin/fasta36 seq.fasta /data/blast/nr/nr > fasta_out.txt

  • BLAST

blastall -p blastp -d /data/blast/nr/nr -i mult_seq.fasta > blast_out.txt

  • PSIBLAST

blastpgn -i seq.fasta -j <#iterations> -h <e-value threshold> -d /data/blast/nr/nr > psiblast_out.txt

  • HHSearch

For the HHSearch tool we used the online server for HHSearch.

Result Statistics

We wrote a script, which shows the distribution of the E-Value and the Identity and also the different aligned sequences. To analyse the overlap between the different methods, we drew a Venn diagram (with http://bioinfogp.cnb.csic.es/tools/venny/index.html). We compared the BLAST, FASTA and Psiblast method (PsiBlast with 3 and 5 runs and E-Value cutoff from 10E-6)


Overlap of the aligned sequences


Comparison ali.png

FASTA found more than 1000 matches, whereas the numbers of results of the blast methods is lower. Therefore, we can see, that FASTA aligns more sequences than blast and therefore, a lot of FASTA hits would be wrong. The two different Psi-Blast variants showed a big agreement by their aligned sequences, and all their sequences were also aligned by FASTA. All sequences, which were aligned by Blast were also found by the FASTA alignment.

We also decided to compare different runs of PsiBlast. We compared PsiBlast with 3 iterations and an e-Value Cutoff of 0.005 and 10E-6 and also two PsiBlast runs with 5 iterations and the same two e-Value cutoffs as before.

Comparison psiblast.png

Psiblast 1: 3 Iterations, E-Value Cutoff: 0.005

Psiblast 2: 5 Iterations, E-Value Cutoff: 0.005

Psiblast 3: 3 Iterations, E-Value Cutoff: 10E-6

Psiblast 4: 5 Iterations, E-Value Cutoff, 10E-6

The differences between the different PsiBlast variants is not that big. Only Psiblast 1 has 6 aligned sequences, which are not shared by the other variants.



TODO:: Seq Id & E-Value



True positive hits

HSSP (Homology-derived Secondary Structure of Proteins) lists proteins which are homologue and have a similar secondary structure. Therefore we use the HSSP alignment to check our results. Therefore we check how much overlap is between HSSP and the other methods. The overlapping sequences are the true positives.

Overlap between HSSP and FASTA
Overlap between HSSP and Blast
Overlap between HSSP and PsiBlast with 3 Iterations and 10E-6 cutoff
Overlap between HSSP and PsiBlast with 5 Iterations and 10E-6 cutoff
Overlap between HSSP and PsiBlast with 3 Iterations and 0.005 cutoff
Overlap between HSSP and psiBlast with 5 Iterations and 0.005 cutoff

Multiple Alignments

  • Cobalt

Download Cobalt from ftp://ftp.ncbi.nlm.nih.gov/pub/cobalt/executables/2.0.1/ (ncbi-cobalt-2.0.1-x64-linux.tar). Uncompress the archive file with tar xfz ncbi-cobalt-2.0.1-x64-linux.tar and change directory to the uncompressed cobalt directoy. Call: ./cobalt -i mult_seq.fasta -norps T > cobalt_out.aln

  • ClustalW

clustalw -infile=mult_seq.fasta > clustalW_out.aln

  • Muscle

muscle -in mult_seq.fasta -out muscle_out.aln -clw

  • T-Coffee

t_coffee -seq mult_seq.fasta

  • T-Coffee (3D)

t_coffee -seq mult_seq.fasta -mode expresso