Difference between revisions of "Sequence Alignments Protocol TSD"

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The results are shown in [[Sequence Alignments TSD|Sequence Alignments TSD]].
  +
 
== Blast ==
 
== Blast ==
<!-- <source lang="bash"> -->
+
<source lang="bash">
 
blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -b 1200 -v 1200 > /mnt/home/student/meiera /1_SeqAli/1_SeqSearch/blastall_1200alis.out
 
blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -b 1200 -v 1200 > /mnt/home/student/meiera /1_SeqAli/1_SeqSearch/blastall_1200alis.out
<!-- </source> -->
+
</source>
 
== PSI-Blast ==
 
== PSI-Blast ==
 
=== Big80 ===
 
=== Big80 ===
  +
<source lang="bash">
time blastpgp -C blastpgp1200_pssmdefault -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_default.out
 
time blastpgp -C blastpgp1200_pssmit2e002 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 2 -h "0.002" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it2e002.out
+
time blastpgp -C blastpgp1200_pssmdefault -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_default.out
time blastpgp -C blastpgp1200_pssmit10e002 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 10 -h "0.002" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it10e002.out
+
time blastpgp -C blastpgp1200_pssmit2e002 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 2 -h "0.002" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it2e002.out
time blastpgp -C blastpgp1200_pssmit10e-10 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 10 -h "10E-10" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it10e-10.out
+
time blastpgp -C blastpgp1200_pssmit10e002 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 10 -h "0.002" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it10e002.out
time blastpgp -C blastpgp1200_pssmit2e-10 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 2 -h "10E-10" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it2e-10.out
+
time blastpgp -C blastpgp1200_pssmit10e-10 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 10 -h "10E-10" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it10e-10.out
  +
time blastpgp -C blastpgp1200_pssmit2e-10 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 2 -h "10E-10" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it2e-10.out
  +
</source>
   
 
=== Big ===
 
=== Big ===
  +
<source lang="bash">
 
PAT="/mnt/home/student/meiera/1_SeqAli/1_SeqSearch"
 
PAT="/mnt/home/student/meiera/1_SeqAli/1_SeqSearch"
 
time blastpgp -R $PAT/blastpgp1200_pssmit2e002 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it2e002.out
 
time blastpgp -R $PAT/blastpgp1200_pssmit2e002 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it2e002.out
Line 17: Line 22:
 
time blastpgp -R $PAT/blastpgp1200_pssmit2e-10 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it2e-10.out
 
time blastpgp -R $PAT/blastpgp1200_pssmit2e-10 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it2e-10.out
 
time blastpgp -R $PAT/blastpgp1200_pssmit10e-10 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it10e-10.out
 
time blastpgp -R $PAT/blastpgp1200_pssmit10e-10 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it10e-10.out
  +
</source>
 
=== Find Unique matches: example ===
+
=== Count unique matches: example ===
  +
<source lang="bash">
 
head -1 m8blastpgp1200_it2e002.out
 
head -1 m8blastpgp1200_it2e002.out
 
grep -n G1RUL9 m8blastpgp1200_it2e002.out
 
grep -n G1RUL9 m8blastpgp1200_it2e002.out
 
tail -n +1233 m8blastpgp1200_it2e002.out | cut -f 2 | wc -l
 
tail -n +1233 m8blastpgp1200_it2e002.out | cut -f 2 | wc -l
 
tail -n +1233 m8blastpgp1200_it2e002.out | uniq -w 44 | wc -l
 
tail -n +1233 m8blastpgp1200_it2e002.out | uniq -w 44 | wc -l
  +
</source>
 
 
== HHblits ==
 
== HHblits ==
  +
<source lang="bash">
 
WD="/mnt/home/student/meiera/1_SeqAli/1_SeqSearch"
 
WD="/mnt/home/student/meiera/1_SeqAli/1_SeqSearch"
 
time hhblits -i $WD/P06865.fasta -o $WD/hhblits_460_P06865.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 460 -B 460 > $WD/hhblits_460_P06865_stdout.log
 
time hhblits -i $WD/P06865.fasta -o $WD/hhblits_460_P06865.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 460 -B 460 > $WD/hhblits_460_P06865_stdout.log
Line 32: Line 39:
 
time hhblits -i $WD/P06865.fasta -n 2 -o $WD/hhblits_2500_P06865_2it.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 2500 -B 2500 > $WD/hhblits_2500_P06865_2_stdout.log
 
time hhblits -i $WD/P06865.fasta -n 2 -o $WD/hhblits_2500_P06865_2it.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 2500 -B 2500 > $WD/hhblits_2500_P06865_2_stdout.log
 
time hhsearch -i $WD/P06865.fasta -o $WD/hhblits_460_P06865.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/pdb70_current_hhm_db -Z 14 -B 14 > $WD/hhblits_pdb_1500_P06865_stdout.log
 
time hhsearch -i $WD/P06865.fasta -o $WD/hhblits_460_P06865.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/pdb70_current_hhm_db -Z 14 -B 14 > $WD/hhblits_pdb_1500_P06865_stdout.log
  +
</source>
 
   
 
==Preparation of outputs==
 
==Preparation of outputs==
   
 
==== Sequence identity and e-values ====
 
==== Sequence identity and e-values ====
  +
<source lang="bash">
 
#for both Blast and HHblits output
 
#for both Blast and HHblits output
 
/mnt/home/student/meiera/bin/1_parseidentity.pl
 
/mnt/home/student/meiera/bin/1_parseidentity.pl
Line 44: Line 52:
 
-o OutFile writes output to fileOUT
 
-o OutFile writes output to fileOUT
 
-h Input is hhblits, default is blast
 
-h Input is hhblits, default is blast
  +
</source>
 
 
==== Uniprot sets for Venn diagrams ====
 
==== Uniprot sets for Venn diagrams ====
  +
<source lang="bash">
 
#for both Blast and HHblits output, also computation of the unique ids for HHblits
 
#for both Blast and HHblits output, also computation of the unique ids for HHblits
 
/mnt/home/student/meiera/bin/1_getUniprotIDS.pl
 
/mnt/home/student/meiera/bin/1_getUniprotIDS.pl
Line 54: Line 63:
 
-h = hhblits input default is set to blastoutput
 
-h = hhblits input default is set to blastoutput
 
-m mappingfile uniprot mappingfile
 
-m mappingfile uniprot mappingfile
  +
</source>
 
 
== Build PDB sets ==
 
== Build PDB sets ==
 
=== Unique identifiers for Blast and HHblits ===
 
=== Unique identifiers for Blast and HHblits ===
  +
<source lang="bash">
 
/mnt/home/student/meiera/bin/1_forPdbMapping.pl
 
/mnt/home/student/meiera/bin/1_forPdbMapping.pl
 
Usage: 1_forPdbMapping.pl [flags] file...
 
Usage: 1_forPdbMapping.pl [flags] file...
Line 90: Line 100:
 
# dont' have to do the special stuff here, the normal one doesn't find any because it's only on uniprot
 
# dont' have to do the special stuff here, the normal one doesn't find any because it's only on uniprot
   
  +
</source>
 
== COPS Parsing ==
 
== COPS Parsing ==
 
The following script will read the COPS clusters (-c) and print out for every classification the members of the cluster that one or several IDs of interest (-i) are contained in. Additionally, if given a list of hits (-hi), it will count the number of hits that fall in the according cluster and the ones that don't and print out that information to a file for plotting e.g. with R.
 
The following script will read the COPS clusters (-c) and print out for every classification the members of the cluster that one or several IDs of interest (-i) are contained in. Additionally, if given a list of hits (-hi), it will count the number of hits that fall in the according cluster and the ones that don't and print out that information to a file for plotting e.g. with R.
Line 96: Line 107:
 
Also be aware that ignoring chains might lead to a merge of clusters. That is if given xxxx als protein of interest and xxxx,A as well was xxxx,B are contained in COPS, the final cluster which is used to calculate how many hits fall into it, will be a combination of the cluster from xxxx,A and the one from xxxx,B. That of course might be overestimating the number of hits that fall into a cluster.
 
Also be aware that ignoring chains might lead to a merge of clusters. That is if given xxxx als protein of interest and xxxx,A as well was xxxx,B are contained in COPS, the final cluster which is used to calculate how many hits fall into it, will be a combination of the cluster from xxxx,A and the one from xxxx,B. That of course might be overestimating the number of hits that fall into a cluster.
   
Sample call: perl parseCOPSClusters.pl -c ~/Desktop/COPS-ChainHierarchy.txt -i ./interestIds -nc -hi ./union.finpdb
+
Sample call: ./parseCOPSClusters.pl -c ~/Desktop/COPS-ChainHierarchy.txt -i ./interestIds -nc -hi ./union.finpdb
   
  +
<source lang="perl">
 
#!/usr/bin/env perl
 
#!/usr/bin/env perl
 
 
Line 332: Line 344:
 
return $id;
 
return $id;
 
}
 
}
  +
  +
</source>
  +
  +
  +
==MSA==
  +
  +
===Dataset creation ===
  +
  +
<source lang="bash">
  +
# Get details for the pdb proteins union
  +
grep -f pdbSets/union.finpdb m8blastpgpBIG_3800*.out > pdbunionBlast
  +
  +
</source>
  +
===Runs===
  +
  +
<source lang="bash">
  +
p="/mnt/home/student/meiera/1_SeqAli/2_MSA"
  +
export PATH=$PATH:/mnt/opt/T-Coffee/bin/
  +
  +
clustalw -align -infile=$p/datasets/msaset_40.fasta -outfile=$p/clustalw_msaset_40.aln
  +
clustalw -align -infile=$p/datasets/msaset_60.fasta -outfile=$p/clustalw_msaset_60.aln
  +
clustalw -align -infile=$p/datasets/msaset_all.fasta -outfile=$p/clustalw_msaset_all.aln
  +
clustalw -infile=$p/datasets/msaset_40.fasta -outfile=$p/clustalw_TESTmsaset_40.aln
  +
  +
muscle -in $p/datasets/msaset_40.fasta -out $p/muscle_msaset_40.msa -quiet
  +
muscle -in $p/datasets/msaset_60.fasta -out $p/muscle_msaset_60.msa -quiet
  +
muscle -in $p/datasets/msaset_all.fasta -out $p/muscle_msaset_all.msa -quiet
  +
  +
t_coffee $p/datasets/msaset_40.fasta -outfile $p/tcdef_40.aln
  +
t_coffee $p/datasets/msaset_60.fasta -outfile $p/tcdef_60.aln
  +
t_coffee $p/datasets/msaset_all.fasta -outfile $p/tcdef_all.aln
  +
t_coffee $p/datasets/msaset_40_struct.fasta -outfile $p/tcdef_40_struct.aln
  +
  +
t_coffee $p/datasets/msaset_40_struct.fasta -method pdb_pair -template_file $p/datasets/msa40struct.temp -outfile $p/tc3d_msa40struct.aln &> $p/tc3d_msa40struct.log
  +
</source>
  +
  +
The template file looks like this:
  +
  +
<source lang="bash">
  +
>sp|P06865|HEXA_HUMAN Beta-hexosaminidase subunit alpha OS=Homo sapiens GN=HEXA PE=1 SV=2 _P_ 2gjxA
  +
>tr|Q06GJ0|Q06GJ0_OSTFU N-acetylglucosaminidase OS=Ostrinia furnacalis PE=1 SV=1 _P_ 3nsnA
  +
>tr|D0VX21|D0VX21_PAESP Beta-hexosaminidase OS=Paenibacillus sp. GN=Hex1 PE=1 SV=1 _P_ 3gh5A
  +
</source>
  +
  +
== Plotting ==
  +
Distribution of hits that full in and out of clusters
  +
  +
  +
<source lang="bash">
  +
library(gplots) #Needs caTools, bitops and a few more. Gives us: barplot2
  +
  +
cbarnames <- 2.5
  +
clwd <- 5
  +
clwdaxis <- 20
  +
ccaxis <- 2
  +
  +
maindat <- read.table('stats2gjx.plotme')
  +
mainmat <- as.matrix(maindat, header=TRUE)
  +
  +
lab <- c("IN", "OUT")
  +
  +
lab <- row.names(mainmat)
  +
len <- length(lab)
  +
  +
method_col <- c("red", "blue")
  +
  +
png("./tsd_inoutdistri.png", height=800, width=800)
  +
# par(xpd=T, mar=par()$mar+c(0,0,0,13))
  +
barplot2(mainmat, beside=TRUE, col=method_col, cex.axis=ccaxis, cex.names=cbarnames, xpd=F, plot.ci=FALSE,cex.main=cbarnames, main="Overlap of union PDB hits with COPS Clusters") #names.arg=c("IN","OUT","IN","OUT","IN","OUT","IN","OUT","IN","OUT")
  +
legend(4,70, lab, fill=method_col, cex=2, ncol=1)
  +
par(mar=c(5, 4, 4, 2) + 0.1) # Restore default clipping rect
  +
dev.off()
  +
  +
</source>
  +
  +
Overlap of PDB hits found by the different methods
  +
  +
  +
  +
<source lang="bash">
  +
library("VennDiagram")
  +
main1<-"Overlap of PDB hits"
  +
sub1<-"BIG database"
  +
  +
hhb <- read.table("data/hhblits.finpdb")
  +
psiit10e002 <- read.table("data/psiblastBIGit10e002.finpdb")
  +
psiit10e10<-read.table("data/psiblastBIGit10e-10.finpdb")
  +
psiit2e002<-read.table("data/psiblastBIGit2e002.finpdb") # psiit2e10<-read.table("data/psiblastBIGit2e-10.finpdb")
  +
  +
psi=list("HHblits"=hhb[,1], "It10, Ev10e-10"=psiit10e10[,1],"It10, Ev002"=psiit10e002[,1],"It2, Ev10e-10/Ev002"=psiit2e002[,1])
  +
  +
  +
venn.diagram(psi,"pdbHitsOverlap.tif", scaled = TRUE,col=c("darkblue", "darkgreen", "orange","darkorchid4"), fill=c("cornflowerblue", "green", "yellow","darkorchid1"),
  +
fontfamily = "serif", fontface = "bold", cat.col = c("darkblue", "darkgreen", "orange","darkorchid4"),cat.cex = 1.5, cat.pos = 0, cat.fontfamily = "serif",
  +
rotation.degree = 200, main=main1, sub=sub1,main.cex=2,sub.cex=1.3,lwd=1,euler.d=2,lty=1, cat.dist=c(0.1,0.1,0.13,0.1))
  +
  +
  +
</source>
  +
e-Value and identity distributions
  +
  +
  +
  +
<source lang="bash">
  +
  +
ccmain <- 1.7
  +
ccaxis <- 2
  +
cclab <- 2
  +
  +
col1 <- "#0000ff"
  +
col2 <- "#ff0000"
  +
col3 <- "#00ff00"
  +
col4 <- "#808080"
  +
col1t <- "#0000FF80"
  +
col2t <- "#FF000080"
  +
col3t <- "#00ff0080"
  +
col4t <- "#80808080"
  +
  +
###Blast
  +
d <- read.table("./temp/blastall1200_IdEvfixed.plot")
  +
d_id <- d$V1
  +
d_ev <- d$V2
  +
  +
#Plot identities
  +
png(width=600, height=600, "tsd_blastallIdent.png")
  +
par(mar=par()$mar+c(0,1,-1,0))
  +
hist(d_id, breaks=100,xlim=c(0,100), main="Blast - Histogram of identities",xlab="% sequence identity in Blast alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
  +
dev.off()
  +
  +
#Plot evalues
  +
png(width=600, height=600, "tsd_blastallEval.png")
  +
par(mar=par()$mar+c(0,1,-1,0))
  +
hist(log(d_ev), breaks=100, main="Blast - Histogram of eValue distribution",xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
  +
dev.off()
  +
  +
###Psi-Blast normal
  +
  +
d1 <- read.table("./temp/blastpgp1200_it2e002_IdEvfixed.plot")
  +
d2 <- read.table("./temp/blastpgp1200_it10e002_IdEvfixed.plot")
  +
d3 <- read.table("./temp/blastpgp1200_it2e-10_IdEvfixed.plot")
  +
d4 <- read.table("./temp/blastpgp1200_it10e-10_IdEvfixed.plot")
  +
  +
d1_id <- d1$V1
  +
d1_ev <- d1$V2
  +
d2_id <- d2$V1
  +
d2_ev <- d2$V2
  +
  +
d3_id <- d3$V1
  +
d3_ev <- d3$V2
  +
d4_id <- d4$V1
  +
d4_ev <- d4$V2
  +
  +
lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
  +
br <- seq(-450,0,5)
  +
  +
png("tsd_psiblastEvals_allfour.png", width=600, height=600)
  +
par(mar=par()$mar+c(0,1,0,0))
  +
hist(log(d1_ev), breaks=br, ylim=c(0,70), xlim=c(-450,0) ,main="PSI-Blast - Histogram of eValue distributions", xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)
  +
mtext("BIG80 database", cex=1.4)
  +
hist(log(d2_ev), breaks=br, ylim=c(0,70),xlim=c(-450,0) ,col=col2t, add=T)
  +
hist(log(d3_ev), breaks=br, ylim=c(0,70),xlim=c(-450,0) ,col=col3t, add=T)
  +
hist(log(d4_ev), breaks=br, ylim=c(0,70),xlim=c(-450,0) ,col=col4t, add=T)
  +
legend("topright", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
  +
dev.off()
  +
  +
  +
lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
  +
br <- seq(0,100,2.5)
  +
  +
png("tsd_psiblastIdents_allfour.png", width=600, height=600)
  +
par(mar=par()$mar+c(0,1,0,0))
  +
hist(d1_id, breaks=br , ylim=c(0,400),main="PSI-Blast - Histogram of identities", xlab="% sequence identity in Blast alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)
  +
mtext("BIG80 database", cex=1.4)
  +
hist(d2_id, breaks=br,col=col2t, add=T)
  +
hist(d3_id, breaks=br, col=col3t, add=T)
  +
hist(d4_id, breaks=br, col=col4t, add=T)
  +
legend("topright", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
  +
dev.off()
  +
  +
  +
  +
###PSI-Blast BIG
  +
d1 <- read.table("./temp/blastpgpBIG_3800_it2e002_IdEvfixed.plot")
  +
d2 <- read.table("./temp/blastpgpBIG_3800_it10e002_IdEvfixed.plot")
  +
d3 <- read.table("./temp/blastpgpBIG_3800_it2e-10_IdEvfixed.plot")
  +
d4 <- read.table("./temp/blastpgpBIG_3800_it10e-10_IdEvfixed.plot")
  +
  +
  +
d1_id <- d1$V1
  +
d1_ev <- d1$V2
  +
d2_id <- d2$V1
  +
d2_ev <- d2$V2
  +
  +
d3_id <- d3$V1
  +
d3_ev <- d3$V2
  +
d4_id <- d4$V1
  +
d4_ev <- d4$V2
  +
  +
lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
  +
br <- seq(-450,0,5)
  +
  +
png("tsd_psiblastBIGEvals_allfour.png", width=600, height=600)
  +
par(mar=par()$mar+c(0,1,0,0))
  +
hist(log(d1_ev), breaks=br, ylim=c(0,260), xlim=c(-450,0) ,main="PSI-Blast - Histogram of eValue distributions", xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)
  +
mtext("BIG database", cex=1.4)
  +
hist(log(d2_ev), breaks=br, ylim=c(0,260),xlim=c(-450,0) ,col=col2t, add=T)
  +
hist(log(d3_ev), breaks=br, ylim=c(0,260),xlim=c(-450,0) ,col=col3t, add=T)
  +
hist(log(d4_ev), breaks=br, ylim=c(0,260),xlim=c(-450,0) ,col=col4t, add=T)
  +
legend("topleft", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
  +
dev.off()
  +
  +
lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
  +
br <- seq(0,100,2.5)
  +
  +
png("tsd_psiblastBIGIdents_allfour.png", width=600, height=600)
  +
par(mar=par()$mar+c(0,1,0,0))
  +
hist(d1_id, breaks=br , ylim=c(0,1100),main="PSI-Blast - Histogram of identities", xlab="% sequence identity in Blast alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)
  +
mtext("BIG database", cex=1.4)
  +
hist(d2_id, breaks=br,col=col2t, add=T)
  +
hist(d3_id, breaks=br, col=col3t, add=T)
  +
hist(d4_id, breaks=br, col=col4t, add=T)
  +
legend("topright", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
  +
dev.off()
  +
  +
  +
###Hblits
  +
d <- read.table("./temp/hhblits_460_P06865_IdEvfixed.plot")
  +
d_id <- d$V1
  +
d_ev <- d$V2
  +
  +
#Plot identities
  +
png(width=600, height=600, "tsd_hhblitsIdent.png")
  +
par(mar=par()$mar+c(0,1,-1,0))
  +
hist(d_id, breaks=100, xlim=c(0,100), main="HHblits - Histogram of identities",xlab="% sequence identity in HHblits alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
  +
dev.off()
  +
  +
#Plot evalues
  +
png(width=600, height=600, "tsd_hhblitsEval.png")
  +
par(mar=par()$mar+c(0,1,-1,0))
  +
hist(log(d_ev), breaks=100, main="HHblits - Histogram of eValue distribution",xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
  +
dev.off()
  +
  +
</source>
  +
Boxplot for MSA gaps
  +
  +
  +
<source lang="bash">
  +
main1<-"Gaps in MSA"
  +
png("testall.png")
  +
boxplot(main=main1,col=c("blue","blue","blue","purple4","purple4","purple4","darkgreen","darkgreen","darkgreen"),names=c("<40%",">60%","Range", "<40%",">60%","Range", "<40%",">60%","Range"), clustal.m40,clustal.m60,clustal.mall,muscle.m40,muscle.m60,muscle.mall,tcoffee.m40,tcoffee.m60,tcoffee.mall,las=2)
  +
legend("topleft",legend=c("ClustalW","Muscle","T-Coffee"),fill=c("blue","purple4","darkgreen"))
  +
dev.off()
  +
</source>

Latest revision as of 15:37, 7 May 2012

The results are shown in Sequence Alignments TSD.

Blast

<source lang="bash">

blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -b 1200 -v 1200 > /mnt/home/student/meiera /1_SeqAli/1_SeqSearch/blastall_1200alis.out

</source>

PSI-Blast

Big80

<source lang="bash"> time blastpgp -C blastpgp1200_pssmdefault -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_default.out time blastpgp -C blastpgp1200_pssmit2e002 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 2 -h "0.002" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it2e002.out time blastpgp -C blastpgp1200_pssmit10e002 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 10 -h "0.002" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it10e002.out time blastpgp -C blastpgp1200_pssmit10e-10 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 10 -h "10E-10" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it10e-10.out time blastpgp -C blastpgp1200_pssmit2e-10 -d /mnt/project/pracstrucfunc12/data/big/big_80 -i /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/P06865.fasta -j 2 -h "10E-10" -v 1200 -b 1200 > /mnt/home/student/meiera/1_SeqAli/1_SeqSearch/blastpgp1200_it2e-10.out </source>

Big

<source lang="bash">

PAT="/mnt/home/student/meiera/1_SeqAli/1_SeqSearch"
time blastpgp -R $PAT/blastpgp1200_pssmit2e002 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it2e002.out
time blastpgp -R $PAT/blastpgp1200_pssmit10e002 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it10e002.out
time blastpgp -R $PAT/blastpgp1200_pssmit2e-10 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it2e-10.out
time blastpgp -R $PAT/blastpgp1200_pssmit10e-10 -d /mnt/project/pracstrucfunc12/data/big/big -i $PAT/P06865.fasta -v 3800 -b 3800 > $PAT/blastpgpBIG_3800_it10e-10.out

</source>

Count unique matches: example

<source lang="bash">

head -1 m8blastpgp1200_it2e002.out
grep -n G1RUL9 m8blastpgp1200_it2e002.out
tail -n +1233 m8blastpgp1200_it2e002.out | cut -f 2 | wc -l
tail -n +1233 m8blastpgp1200_it2e002.out | uniq -w 44 | wc -l

</source>

HHblits

<source lang="bash">

WD="/mnt/home/student/meiera/1_SeqAli/1_SeqSearch"
time hhblits -i $WD/P06865.fasta -o $WD/hhblits_460_P06865.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 460 -B 460 > $WD/hhblits_460_P06865_stdout.log
time hhblits -i $WD/P06865.fasta -n 10 -o $WD/hhblits_460_P06865_10it.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 460 -B 460 > $WD/hhblits_460_P06865_10_stdout.log
time hhblits -i $WD/P06865.fasta -n 10 -o $WD/hhblits_2500_P06865_10it.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 2500 -B 2500 > $WD/hhblits_2500_P06865_10_stdout.log
time hhblits -i $WD/P06865.fasta -n 2 -o $WD/hhblits_460_P06865_2it.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 460 -B 460 > $WD/hhblits_460_P06865_2_stdout.log
time hhblits -i $WD/P06865.fasta -n 2 -o $WD/hhblits_2500_P06865_2it.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -Z 2500 -B 2500 > $WD/hhblits_2500_P06865_2_stdout.log
time hhsearch -i $WD/P06865.fasta -o $WD/hhblits_460_P06865.hhr -d /mnt/project/pracstrucfunc12/data/hhblits/pdb70_current_hhm_db -Z 14 -B 14 > $WD/hhblits_pdb_1500_P06865_stdout.log

</source>

Preparation of outputs

Sequence identity and e-values

<source lang="bash">

#for both Blast and HHblits output
/mnt/home/student/meiera/bin/1_parseidentity.pl
Usage: 1_parseidentity.pl [flags] file...
flags:
   -i Infile   reads the input from fileIN 
   -o OutFile  writes output to fileOUT 
   -h	Input is hhblits, default is blast

</source>

Uniprot sets for Venn diagrams

<source lang="bash">

#for both Blast and HHblits output, also computation of the unique ids for HHblits
 /mnt/home/student/meiera/bin/1_getUniprotIDS.pl
Usage: 1_getUniprotIDS.pl [flags] file...
flags:
   -i Infile   reads the input from fileIN 
   -o OutFile  writes uniprot identifiers to fileOUT 
   -h = hhblits input default is set to blastoutput
   -m mappingfile   uniprot mappingfile

</source>

Build PDB sets

Unique identifiers for Blast and HHblits

<source lang="bash">

/mnt/home/student/meiera/bin/1_forPdbMapping.pl
Usage: 1_forPdbMapping.pl [flags] file...
flags:
   -i Infile   reads the input from fileIN 
   -o OutFile  writes uniprot identifiers to fileOUT 
   -h = hhblits input, default is set to blastoutput


#Use uniprot.org's online mapping service on the .pdbmapping files and save the results to .mapped files
cut -f 2 psiblastBIGit10e002.mapped | tr '[A-Z]' '[a-z]' | sort | uniq | grep -P "\w{4}" >  psiblastBIGit10e002.finpdb1
grep -P "\w{4}_" psiblastBIGit10e002.pdbmapping --only-matching | sed "s/_//" >  psiblastBIGit10e002.finpdb2
cat psiblastBIGit10e002.finpdb2 psiblastBIGit10e002.finpdb1 | sort | uniq > psiblastBIGit10e002.finpdb
cut -f 2  psiblastBIGit10e-10.mapped | tr '[A-Z]' '[a-z]' | sort | uniq | grep -P "\w{4}" >   psiblastBIGit10e-10.finpdb1
grep -P "\w{4}_" psiblastBIGit10e-10.pdbmapping --only-matching | sed "s/_//" >  psiblastBIGit10e-10.finpdb2
cat psiblastBIGit10e-10.finpdb1 psiblastBIGit10e-10.finpdb2 | sort | uniq > psiblastBIGit10e-10.finpdb
cut -f 2 psiblastBIGit2e002.mapped | tr '[A-Z]' '[a-z]' | sort | uniq | grep -P "\w{4}" >  psiblastBIGit2e002.finpdb1
grep -P "\w{4}_" psiblastBIGit2e002.pdbmapping --only-matching | sed "s/_//" >  psiblastBIGit2e002.finpdb2
cat psiblastBIGit2e002.finpdb1 psiblastBIGit2e002.finpdb2 | sort | uniq > psiblastBIGit2e002.finpdb
cut -f 2 psiblastBIGit2e-10.mapped | tr '[A-Z]' '[a-z]' | sort | uniq | grep -P "\w{4}" >  psiblastBIGit2e-10.finpdb1
grep -P "\w{4}_" psiblastBIGit2e-10.pdbmapping --only-matching | sed "s/_//" >  psiblastBIGit2e-10.finpdb2
cat psiblastBIGit2e-10.finpdb1 psiblastBIGit2e-10.finpdb2 | sort | uniq > psiblastBIGit2e-10.finpdb


cut -f 2 hhblits__460.mapped | tr '[A-Z]' '[a-z]' | sort | uniq | grep -P "\w{4}" > hhbtemp
sed "s/_\w//g" hhblits_pdb_1500.pdbmapping > hhbtemp2
cat hhbtemp hhbtemp2 | sed "s/\s//g" |  sort | uniq > hhblits.finpdb
rm -f hhbtemp hhbtemp2
# dont' have to do the special stuff here, the normal one doesn't find any because it's only on uniprot

</source>

COPS Parsing

The following script will read the COPS clusters (-c) and print out for every classification the members of the cluster that one or several IDs of interest (-i) are contained in. Additionally, if given a list of hits (-hi), it will count the number of hits that fall in the according cluster and the ones that don't and print out that information to a file for plotting e.g. with R. Note that you can ignore chains with the '-nc' option.

Also be aware that ignoring chains might lead to a merge of clusters. That is if given xxxx als protein of interest and xxxx,A as well was xxxx,B are contained in COPS, the final cluster which is used to calculate how many hits fall into it, will be a combination of the cluster from xxxx,A and the one from xxxx,B. That of course might be overestimating the number of hits that fall into a cluster.

Sample call: ./parseCOPSClusters.pl -c ~/Desktop/COPS-ChainHierarchy.txt -i ./interestIds -nc -hi ./union.finpdb

<source lang="perl">

#!/usr/bin/env perl

use strict;
use warnings;
use sigtrap;
use autodie;
use diagnostics;
use Pod::Usage;
use Getopt::Long qw(:config require_order);
use 5.010;    #For smart match, which makes this script unbelievably slow, but I'm lazy :)

##Program variables
my %clusters;
my @clusterOrder;
my @interestIDs = ();
my %interestIDLocations;
my @hitsIDs = ();

##Argument
my $copsPath     = '/mnt/project/pracstrucfunc12/data/COPS/COPS-ChainHierarchy.txt';
my $nochain      = 0;
my $interestPath = './interestIds';
my $hitsPath     = './union.finpdb';

########################
###Argument Handling####
########################
my $result = GetOptions(
    'help|h|?'     => sub { pod2usage( -verbose => 1 ); },
    'man|m'        => sub { pod2usage( -verbose => 2 ); },
    'c|cops=s'     => \$copsPath,
    'nc|nocain'    => \$nochain,
    'i|interest=s' => \$interestPath,
    'hi|hits=s'    => \$hitsPath
) or pod2usage( -verbose => 1 ) && exit;

#Read IDs to look out for
my $fhi;
open( $fhi, '<', $interestPath );
while ( my $line = <$fhi> )
{
    if ( $line =~ m/(\w{4},\w)/ || $line =~ m/(\w{4})/ )
    {
        my $iid = lc($1);
        push( @interestIDs, $iid );
        $interestIDLocations{$iid} = {};
    }
}

close($fhi);

#Read ids that were found by prediction method(s)
my $fhh;
open($fhh, '<', $hitsPath);
while(my $line =<$fhh>)
{
    if($line =~ m/^(\w{4})$/)
    {
        push(@hitsIDs, lc($1));
    }
}
close($fhh);

print "Found # hits: " . scalar(@hitsIDs) . "\n";

#Parse the COPS structure
my $fh;
open( $fh, '<', $copsPath );
while ( my $line = <$fh> )
{
    if ( $line =~ m/^Chain (.+)$/ )
    {
        #Header line found. Inititialize the cluster hash
        my @matches = $1 =~ m/\w+/g;
        for my $clusterName (@matches)
        {
            $clusters{$clusterName} = {};
        }

        @clusterOrder = @matches;
    }
    elsif ( $line =~ m/\w{4},/ )
    {
        #Cluster info line
        my @matches = $line =~ m/(\w{4},\w)/g;
        my $currentId = shift(@matches);

        #         print "current id is $currentId\n";
        $currentId = sanitizePdbIDs($currentId);

        my $count = 0;

        for my $representative (@matches)
        {
            $representative = sanitizePdbIDs($representative);

            my $currentCluster = $clusterOrder[ $count++ ];

            #             print "currentCluster is $currentCluster\n";

            if ( !exists $clusters{$currentCluster} || !defined $clusters{$currentCluster} )
            {
                print "this should not have happened :)\n";
                next;
            }
            if ( !exists $clusters{$currentCluster}{$representative} )
            {

                #Inititialize the cluster with the representative
                $clusters{$currentCluster}{$representative} = [$representative];
            }

            #Add the current pdb id

            if ( !( $currentId ~~ @{ $clusters{$currentCluster}{$representative} } ) )
            {

                #This case can only occur when we do not use the chains
                push( @{ $clusters{$currentCluster}{$representative} }, $currentId );
            }

            if ( $currentId ~~ @interestIDs )
            {
                if(!defined $interestIDLocations{$currentId}{$currentCluster})
                {
                    $interestIDLocations{$currentId}{$currentCluster}= ();
                }
                push(@{$interestIDLocations{$currentId}{$currentCluster} }, $representative);
            }
        }
    }
}
close($fh);


#Do some maybe interesting output
for my $intId (@interestIDs)
{
    printClustersForId($intId);
}

sub printClustersForId
{
    my $id = shift;
    
    my @allIn = ();
    my @allOut = ();
    
    for my $cluster ( sort(keys( $interestIDLocations{$id} ) ) )
    {        
        my %clusterMembersMerged = ();
        
        print "MEMBERS: $id -- $cluster : ";
        my @currentRepresentatives = @{$interestIDLocations{$id}{$cluster}};
        
        for my $currentRepresentative (@currentRepresentatives)
        {
            my @clusterMembers        = @{ $clusters{$cluster}{$currentRepresentative} };

            for my $member (@clusterMembers)
            {
#                 print "$member ";
                $clusterMembersMerged{$member} = ;
            }
#             print "\n"; 
        }
        
        my @clusterMembersMergedArr= keys(%clusterMembersMerged);
        
        for my $e (@clusterMembersMergedArr)
        {
            print $e." ";
        }
        print "\n";
        
        
        my $hitsIn = 0;
        my $hitsOut = 0;
        
        print "OVERLAPS: $id -- $cluster : ";
        for my $hit (@hitsIDs)
        {
            if($hit ~~ @clusterMembersMergedArr)
            {
                $hitsIn++;
            }
            else
            {
                $hitsOut++;
            }
        }
        
        print "In: $hitsIn, Out: $hitsOut\n";     
        push(@allIn, $hitsIn);
        push(@allOut, $hitsOut);
    }
    
    #Write Out In/Out over Cluster stats for plotting in R
    my $fho;
    open($fho, '>', "stats$id.plotme");
    for my $cluster (sort (keys( $interestIDLocations{$id})))
    {
        print $fho "\t$cluster";
    }
    print $fho "\n";
    
    print $fho "IN";
    for my $elem (@allIn)
    {
        print $fho "\t$elem";
    }
    print $fho "\n";
    
    print $fho "OUT";
    for my $elem (@allOut)
    {
        print $fho "\t$elem";
    }
    print $fho "\n";
    
    close($fho);    
}



sub sanitizePdbIDs
{
    my $id = shift;
    if ($nochain)
    {
        $id = substr( $id, 0, 4, );
    }
    return $id;
}

</source>


MSA

Dataset creation

<source lang="bash">

# Get details for the pdb proteins union
grep -f  pdbSets/union.finpdb  m8blastpgpBIG_3800*.out > pdbunionBlast

</source>

Runs

<source lang="bash">

p="/mnt/home/student/meiera/1_SeqAli/2_MSA"
export PATH=$PATH:/mnt/opt/T-Coffee/bin/
clustalw -align -infile=$p/datasets/msaset_40.fasta -outfile=$p/clustalw_msaset_40.aln
clustalw -align -infile=$p/datasets/msaset_60.fasta -outfile=$p/clustalw_msaset_60.aln
clustalw -align -infile=$p/datasets/msaset_all.fasta -outfile=$p/clustalw_msaset_all.aln
clustalw -infile=$p/datasets/msaset_40.fasta -outfile=$p/clustalw_TESTmsaset_40.aln
muscle -in $p/datasets/msaset_40.fasta -out $p/muscle_msaset_40.msa -quiet
muscle -in $p/datasets/msaset_60.fasta -out $p/muscle_msaset_60.msa -quiet
muscle -in $p/datasets/msaset_all.fasta -out $p/muscle_msaset_all.msa -quiet

t_coffee $p/datasets/msaset_40.fasta -outfile $p/tcdef_40.aln t_coffee $p/datasets/msaset_60.fasta -outfile $p/tcdef_60.aln t_coffee $p/datasets/msaset_all.fasta -outfile $p/tcdef_all.aln t_coffee $p/datasets/msaset_40_struct.fasta -outfile $p/tcdef_40_struct.aln

t_coffee $p/datasets/msaset_40_struct.fasta -method pdb_pair -template_file $p/datasets/msa40struct.temp -outfile $p/tc3d_msa40struct.aln &> $p/tc3d_msa40struct.log </source>

The template file looks like this:

<source lang="bash"> >sp|P06865|HEXA_HUMAN Beta-hexosaminidase subunit alpha OS=Homo sapiens GN=HEXA PE=1 SV=2 _P_ 2gjxA >tr|Q06GJ0|Q06GJ0_OSTFU N-acetylglucosaminidase OS=Ostrinia furnacalis PE=1 SV=1 _P_ 3nsnA >tr|D0VX21|D0VX21_PAESP Beta-hexosaminidase OS=Paenibacillus sp. GN=Hex1 PE=1 SV=1 _P_ 3gh5A </source>

Plotting

Distribution of hits that full in and out of clusters


<source lang="bash">

library(gplots)     #Needs caTools, bitops and a few more. Gives us: barplot2

cbarnames <- 2.5
clwd <- 5
clwdaxis <- 20
ccaxis <- 2

maindat <- read.table('stats2gjx.plotme')
mainmat <- as.matrix(maindat, header=TRUE)

lab <- c("IN", "OUT")

 lab <- row.names(mainmat)
        len <- length(lab)

        method_col <- c("red", "blue")

png("./tsd_inoutdistri.png", height=800, width=800)
# par(xpd=T, mar=par()$mar+c(0,0,0,13))
barplot2(mainmat, beside=TRUE, col=method_col, cex.axis=ccaxis, cex.names=cbarnames, xpd=F, plot.ci=FALSE,cex.main=cbarnames, main="Overlap of union PDB hits with COPS Clusters")        #names.arg=c("IN","OUT","IN","OUT","IN","OUT","IN","OUT","IN","OUT")
legend(4,70, lab, fill=method_col, cex=2, ncol=1)
par(mar=c(5, 4, 4, 2) + 0.1)    # Restore default clipping rect
dev.off()

</source>

Overlap of PDB hits found by the different methods


<source lang="bash">

library("VennDiagram")
main1<-"Overlap of PDB hits"
sub1<-"BIG database"

hhb <- read.table("data/hhblits.finpdb")
psiit10e002 <- read.table("data/psiblastBIGit10e002.finpdb")
psiit10e10<-read.table("data/psiblastBIGit10e-10.finpdb")
psiit2e002<-read.table("data/psiblastBIGit2e002.finpdb")    # psiit2e10<-read.table("data/psiblastBIGit2e-10.finpdb")

psi=list("HHblits"=hhb[,1], "It10, Ev10e-10"=psiit10e10[,1],"It10, Ev002"=psiit10e002[,1],"It2, Ev10e-10/Ev002"=psiit2e002[,1])


venn.diagram(psi,"pdbHitsOverlap.tif", scaled = TRUE,col=c("darkblue", "darkgreen", "orange","darkorchid4"), fill=c("cornflowerblue", "green", "yellow","darkorchid1"), 
             fontfamily = "serif", fontface = "bold", cat.col = c("darkblue", "darkgreen", "orange","darkorchid4"),cat.cex = 1.5, cat.pos = 0, cat.fontfamily = "serif", 
             rotation.degree = 200, main=main1, sub=sub1,main.cex=2,sub.cex=1.3,lwd=1,euler.d=2,lty=1, cat.dist=c(0.1,0.1,0.13,0.1)) 


</source> e-Value and identity distributions


<source lang="bash">

ccmain <- 1.7
ccaxis <- 2
cclab <- 2

col1 <- "#0000ff"
col2 <- "#ff0000"
col3 <- "#00ff00"
col4 <- "#808080"
col1t <- "#0000FF80"
col2t <- "#FF000080"
col3t <- "#00ff0080"
col4t <- "#80808080"

###Blast
d <- read.table("./temp/blastall1200_IdEvfixed.plot")
d_id <- d$V1
d_ev <- d$V2

#Plot identities
png(width=600, height=600, "tsd_blastallIdent.png")
par(mar=par()$mar+c(0,1,-1,0))
hist(d_id, breaks=100,xlim=c(0,100), main="Blast - Histogram of identities",xlab="% sequence identity in Blast alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
dev.off()

#Plot evalues
png(width=600, height=600, "tsd_blastallEval.png")
par(mar=par()$mar+c(0,1,-1,0))
hist(log(d_ev), breaks=100, main="Blast - Histogram of eValue distribution",xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
dev.off()

###Psi-Blast normal

d1 <- read.table("./temp/blastpgp1200_it2e002_IdEvfixed.plot")
d2 <- read.table("./temp/blastpgp1200_it10e002_IdEvfixed.plot")
d3 <- read.table("./temp/blastpgp1200_it2e-10_IdEvfixed.plot")
d4 <- read.table("./temp/blastpgp1200_it10e-10_IdEvfixed.plot")

d1_id <- d1$V1
d1_ev <- d1$V2
d2_id <- d2$V1
d2_ev <- d2$V2

d3_id <- d3$V1
d3_ev <- d3$V2
d4_id <- d4$V1
d4_ev <- d4$V2

lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
br <- seq(-450,0,5)

png("tsd_psiblastEvals_allfour.png", width=600, height=600)
par(mar=par()$mar+c(0,1,0,0))
hist(log(d1_ev), breaks=br, ylim=c(0,70), xlim=c(-450,0) ,main="PSI-Blast - Histogram of eValue distributions", xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)     
mtext("BIG80 database", cex=1.4)
hist(log(d2_ev), breaks=br, ylim=c(0,70),xlim=c(-450,0) ,col=col2t, add=T)
hist(log(d3_ev), breaks=br, ylim=c(0,70),xlim=c(-450,0) ,col=col3t, add=T)
hist(log(d4_ev), breaks=br, ylim=c(0,70),xlim=c(-450,0) ,col=col4t, add=T)
legend("topright", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
dev.off()


lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
br <- seq(0,100,2.5)

png("tsd_psiblastIdents_allfour.png", width=600, height=600)
par(mar=par()$mar+c(0,1,0,0))
hist(d1_id, breaks=br , ylim=c(0,400),main="PSI-Blast - Histogram of identities", xlab="% sequence identity in Blast alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)     
mtext("BIG80 database", cex=1.4)
hist(d2_id, breaks=br,col=col2t, add=T)
hist(d3_id, breaks=br, col=col3t, add=T)
hist(d4_id, breaks=br, col=col4t, add=T)
legend("topright", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
dev.off()



###PSI-Blast BIG
d1 <- read.table("./temp/blastpgpBIG_3800_it2e002_IdEvfixed.plot")
d2 <- read.table("./temp/blastpgpBIG_3800_it10e002_IdEvfixed.plot")
d3 <- read.table("./temp/blastpgpBIG_3800_it2e-10_IdEvfixed.plot")
d4 <- read.table("./temp/blastpgpBIG_3800_it10e-10_IdEvfixed.plot")


d1_id <- d1$V1
d1_ev <- d1$V2
d2_id <- d2$V1
d2_ev <- d2$V2

d3_id <- d3$V1
d3_ev <- d3$V2
d4_id <- d4$V1
d4_ev <- d4$V2

lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
br <- seq(-450,0,5)

png("tsd_psiblastBIGEvals_allfour.png", width=600, height=600)
par(mar=par()$mar+c(0,1,0,0))
hist(log(d1_ev), breaks=br, ylim=c(0,260), xlim=c(-450,0) ,main="PSI-Blast - Histogram of eValue distributions", xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)     
mtext("BIG database", cex=1.4)
hist(log(d2_ev), breaks=br, ylim=c(0,260),xlim=c(-450,0) ,col=col2t, add=T)
hist(log(d3_ev), breaks=br, ylim=c(0,260),xlim=c(-450,0) ,col=col3t, add=T)
hist(log(d4_ev), breaks=br, ylim=c(0,260),xlim=c(-450,0) ,col=col4t, add=T)
legend("topleft", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
dev.off()

lab = c("2 iterations, e002", "10 iterations, e002", "2 Iterations, 10e-10", "10 Iterations, 10e-10")
br <- seq(0,100,2.5)

png("tsd_psiblastBIGIdents_allfour.png", width=600, height=600)
par(mar=par()$mar+c(0,1,0,0))
hist(d1_id, breaks=br , ylim=c(0,1100),main="PSI-Blast - Histogram of identities", xlab="% sequence identity in Blast alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1t)     
mtext("BIG database", cex=1.4)
hist(d2_id, breaks=br,col=col2t, add=T)
hist(d3_id, breaks=br, col=col3t, add=T)
hist(d4_id, breaks=br, col=col4t, add=T)
legend("topright", lab, fill=c(col1t, col2t, col3t, col4t), cex=1.6, ncol=1)
dev.off()


###Hblits
d <- read.table("./temp/hhblits_460_P06865_IdEvfixed.plot")
d_id <- d$V1
d_ev <- d$V2

#Plot identities
png(width=600, height=600, "tsd_hhblitsIdent.png")
par(mar=par()$mar+c(0,1,-1,0))
hist(d_id, breaks=100, xlim=c(0,100), main="HHblits - Histogram of identities",xlab="% sequence identity in HHblits alignment", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
dev.off()

#Plot evalues
png(width=600, height=600, "tsd_hhblitsEval.png")
par(mar=par()$mar+c(0,1,-1,0))
hist(log(d_ev), breaks=100, main="HHblits - Histogram of eValue distribution",xlab="log(eValue)", cex.axis=ccaxis, cex.main=ccmain, cex.lab=cclab, col=col1)
dev.off()

</source> Boxplot for MSA gaps


<source lang="bash">

main1<-"Gaps in MSA"
png("testall.png")
boxplot(main=main1,col=c("blue","blue","blue","purple4","purple4","purple4","darkgreen","darkgreen","darkgreen"),names=c("<40%",">60%","Range", "<40%",">60%","Range", "<40%",">60%","Range"), clustal.m40,clustal.m60,clustal.mall,muscle.m40,muscle.m60,muscle.mall,tcoffee.m40,tcoffee.m60,tcoffee.mall,las=2)
legend("topleft",legend=c("ClustalW","Muscle","T-Coffee"),fill=c("blue","purple4","darkgreen"))
dev.off()

</source>