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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 <code> real 11m30.762s<br> user 3m11.440s<br> sys 0m12.250s<br> </code> ====Results==== ===FASTA=== ====Installation==== * Used Virtual Box with Linux. * Download fasta3.tar.gz from ftp://ftp.ebi.ac.uk/pub/software/unix/fasta/ * Unzip the archive * Build it with: make -f /apps/fasta/make/Makefile.linux64 all ====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 <code> real 10m13.878s <br> user 7m26.270s <br> sys 0m20.230s <br> </code> ====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' <code> real 37m56.447s<br> user 14m27.620s<br> sys 0m54.620s<br> </code> =====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' <code> real 37m41.487s <br> user 14m42.850s <br> sys 0m52.370s <br> </code> =====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' <code> real 62m22.175s <br> user 26m25.410s <br> sys 1m20.700s <br> </code> =====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' <code> real 61m59.284s<br> user 25m55.920s<br> sys 1m21.620s<br> </code> ====Results==== ===HHSearch=== ====Installation==== =====Preparing the HHM-Database===== * Download pdb70_29May10.hhm.tar.gz from ftp://ftp.tuebingen.mpg.de/pub/protevo/HHsearch/databases/ * Unzip the archive * Make a database: cat *.hhm >> pdb70.db * Move the db to an appropriate directory: sudo mv pdb70.db ../pdb70.db =====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:<br> <code> my $psipreddir="/apps/psipred_2.5"; <br> my $ncbidir="/apps/blast_old/bin"; <br> my $perl="/apps/bin"; <br> my $dummydb="/home/student/tmp"; <br> </code> * 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 <code> real 8m33.171s <br> user 5m14.530s <br> sys 0m3.510s <br> </code> =====Parameterset 2===== alignblast reference_psi_e10E-6_i3.blast reference_psi_e10E-6_i3.a3m <br> addpsipred /home/student/workspace/reference_psi_e10E-6_i3.a3m <br> time hhsearch -i reference_psi_e10E-6_i3.a3m -d /data/hmm/pdb70.db -o reference_psi_e10E-6_i3.hhsearch <br> <code> real 16m27.258s <br> user 7m47.220s <br> sys 0m6.290s <br> </code> 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 <br> addpsipred /home/student/workspace/reference_psi_e005_i3.a3m <br> time hhsearch -i reference_psi_e005_i3.a3m -d /data/hmm/pdb70.db -o reference_psi_e005_i3.hhsearch <br> <code> real 16m7.216s<br> user 7m41.840s<br> sys 0m5.570s<br> </code> =====Parameterset 4===== alignblast reference_psi_e005_i5.blast reference_psi_e005_i5.blast.a3m <br> addpsipred /home/student/workspace/reference_psi_e005_i5.blast.a3m <br> time hhsearch -i reference_psi_e005_i5.blast.a3m -d /data/hmm/pdb70.db -o reference_psi_e005_i5.blast.hhsearch <br> <code> real 7m49.907s<br> user 7m15.310s<br> sys 0m4.320s<br> </code> =====Parameterset 5===== alignblast reference_psi_e10E-6_i5.blast reference_psi_e10E-6_i5.a3m<br> addpsipred /home/student/workspace/reference_psi_e10E-6_i5.a3m<br> time hhsearch -i reference_psi_e10E-6_i5.a3m -d /data/hmm/pdb70.db -o reference_psi_e10E-6_i5.hhsearch<br> <code> real 8m10.730s<br> user 7m33.190s<br> sys 0m5.390s<br> </code> ===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 was run with a dataset of the PDB. BLAST was run with the a dataset of the NR database. NR itself is a mixture of SwissProt, RefSeq, PIR, PRF, PDB and GenBank CDS translations. HSSP itself contains only references to SwissProt entries. Therefore it was necessary to get the cross-references of the Swissprot entries in HSSP to the other databases. For this purpose we created a java-tool (only runable with linux).

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