Task 2 - Alignments with PAH Reference

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Task 2 - Alignments with PAH Reference

Sequence Searches

BLAST

Running

time sudo blastall -p blastp -d '/data/blast/nr/nr' -i ./reference.fasta -o './reference.blast' -b 500

real 11m30.762s
user 3m11.440s
sys 0m12.250s

Results

FASTA

Installation

  • Used Virtual Box with Linux.

Running

time ./fasta36 /home/student/reference.fasta /data/nr/nr

interactive:

  • Enter filename for results []: /home/student/reference.fasta_search
  • How many scores do you want to see: 500
  • More scores? 0
  • Display alignments also? (y/n) [n] y
  • number of alignments [500]? 500

real 10m13.878s
user 7m26.270s
sys 0m20.230s

Results

PSI-BLAST

Running

Parameterset 1

time blastpgp -d '/data/nr/nr' -i './reference.fasta' -o './reference_psi_e10E-6_i3.blast' -h 10E-6 -j 3 -C './reference_i3_e10E-6.chk'

real 37m56.447s
user 14m27.620s
sys 0m54.620s

Parameterset 2

time blastpgp -d '/data/nr/nr' -i './reference.fasta' -o './reference_psi_e005_i3.blast' -h 0.005 -j 3 -C './reference_i3_e005.chk'

real 37m41.487s
user 14m42.850s
sys 0m52.370s

Parameterset 3

time blastpgp -d '/data/nr/nr' -i './reference.fasta' -o './reference_psi_e005_i5.blast' -h 0.005 -j 5 -C './reference_i5_e005.chk'

real 62m22.175s
user 26m25.410s
sys 1m20.700s

Parameterset 4

time blastpgp -d '/data/nr/nr' -i './reference.fasta' -o './reference_psi_e10E-6_i5.blast' -h 10E-6 -j 5 -C './reference_i5_e10E-6.chk'

real 61m59.284s
user 25m55.920s
sys 1m21.620s

Results

HHSearch

Installation

Preparing the HHM-Database
Configure HHSearch-Tools

In the manual of HHSearch it was adviced to add the information of the secondary structure to the multiple alignment used for the query. Therefore it was necessary to run the addpsipred script of HHSearch. This script was not configured in the virtual box. Several parameters have to be adjusted.

  • Changes in /apps/bin/addpsipred:

my $psipreddir="/apps/psipred_2.5";
my $ncbidir="/apps/blast_old/bin";
my $perl="/apps/bin";
my $dummydb="/home/student/tmp";

  • Copy /apps/bin/reformat to /apps/bin/reformat.pl


Running

Parameterset 1

time hhsearch -i ./reference.fasta -d /data/hmm/pdb70.db -b 500 -o ./reference_simple.hhsearch

real 8m33.171s
user 5m14.530s
sys 0m3.510s

Parameterset 2

alignblast reference_psi_e10E-6_i3.blast reference_psi_e10E-6_i3.a3m
addpsipred /home/student/workspace/reference_psi_e10E-6_i3.a3m
time hhsearch -i reference_psi_e10E-6_i3.a3m -d /data/hmm/pdb70.db -o reference_psi_e10E-6_i3.hhsearch

real 16m27.258s
user 7m47.220s
sys 0m6.290s

To run HHSearch with the profile of a PSI-BLAST search the output of PSI-BLAST has to be converted to a multiple alignment by the script alignblast. The performance of HHSearch can be increases by adding the information of the secondary structure to the multiple alignment. This can be done with the script addspipred (uses the secondary structure prediction of PSIPRED).

Parameterset 3

alignblast reference_psi_e005_i3.blast reference_psi_e005_i3.a3m
addpsipred /home/student/workspace/reference_psi_e005_i3.a3m
time hhsearch -i reference_psi_e005_i3.a3m -d /data/hmm/pdb70.db -o reference_psi_e005_i3.hhsearch

real 16m7.216s
user 7m41.840s
sys 0m5.570s

Parameterset 4

alignblast reference_psi_e005_i5.blast reference_psi_e005_i5.blast.a3m
addpsipred /home/student/workspace/reference_psi_e005_i5.blast.a3m
time hhsearch -i reference_psi_e005_i5.blast.a3m -d /data/hmm/pdb70.db -o reference_psi_e005_i5.blast.hhsearch

real 7m49.907s
user 7m15.310s
sys 0m4.320s

Parameterset 5

alignblast reference_psi_e10E-6_i5.blast reference_psi_e10E-6_i5.a3m
addpsipred /home/student/workspace/reference_psi_e10E-6_i5.a3m
time hhsearch -i reference_psi_e10E-6_i5.a3m -d /data/hmm/pdb70.db -o reference_psi_e10E-6_i5.hhsearch

real 8m10.730s
user 7m33.190s
sys 0m5.390s

Comparing the Results

HSSP - Some Positives

Getting the entry of PAH from HSSP http://mrs.cmbi.ru.nl/mrs-5/entry?db=hssp&id=2pah&q=phenylalanine%20hydroxylase

HSSP - More Positives

hhsearch is run with a pdb-set. for blast was nr used. nr contains swissprot, refseq, PIR, PRF, PDB and GenBank CDS translations entries. hssp contains only swissprot entries. That's why a mapping of the swissprot-entries and the other databases is necessary. For this purpose we created a java-tool:

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