This document describes steps in a bioinformatics analysis pipeline that uses BLAST and CLUSTALW. The pipeline takes a protein sequence from KEGG identified by its ID (hsa:6469), runs BLAST to find similar sequences, parses the BLAST output, runs CLUSTALW for multiple sequence alignment, and outputs the results. Ruby scripts are used to implement each step via calling REST and SOAP services for BLAST and CLUSTALW.

1. 2010/06/10
kaneko.satoko(at)ocha.ac.jp

2. BLAST CLUSTALW

3. BLAST CLUSTALW
TogoWS REST SOAP (step )
(11):E‐value

4. (1)
blast_clustalw.rb
blast_clustalw.rb
$ ruby blast_clustalw.rb hsa:6469
( has:6469 blast + clustalw
blast_clustalw.rb
step10.rb
step20.rb Blastp
step30.rb Blastp ID E‐value
step40.rb ID E‐value E‐value
step50.rb
step60.rb ClustalW mulSple alignment

5. (2) step10.rb
k.step10.txt
‐ hsa:6469 ( ID kegg_id )
‐blast_clustalw.rb 8‐12 get_aaseq
‐ last_clustalw.rb
b get_aaseq 79
#!/usr/bin/env ruby
require ‘open‐uri’ # 8 open URL REST
# kegg_id
kegg_id = "hsa:6469";
# kegg_id aaseq
aaseq = open("hap://togows.dbcls.jp/entry/kegg‐genes/" + kegg_id + "/aaseq").read
#REST hap://togows.dbcls.jp/entry/kegg‐genes/has:6469/aaseq
# aaseq
# aaseq k.step10.txt
out = open("k.step10.txt","w")
out.puts aaseq
out.close
CotEditor

6. (3) step20.rb
Sonic Hedgehog query BLAST
‐blast_clustalw.rb 15‐25
‐ blast_clustalw.rb 81
#!/usr/bin/env ruby
require 'soap/wsdlDriver'
# step10.rb aaseq
aaseq = File.read("k.step10.txt”)
# SOAP blast result_blast
wsdl_blast = "hap://togows.dbcls.jp/soap/wsdl/ddbj_blast.wsdl"
serv_blast = SOAP::WSDLDriverFactory.new(wsdl_blast).create_rpc_driver
program = "blastp"
database = "SWISS"
result_blast = serv_blast.DdbjBlastSearchSimple(program,database,aaseq)
# result_blast k.step20.txt
out = open("k.step20.txt","w")
out.puts result_blast
out.close

7. (4) step30.rb
BLAST clustalw
‐ blast_clustalw.rb 27 47 84
#!/usr/bin/env ruby
# step20.rb blast result_blast
result_blast = File.read("k.step20.txt")
# result_blast spid_hash
spid_hash = Hash.new #
# blast line
result_blast.each_line do |line|
# blast sp
if line =~ /^sp|(.+)|(.+)s+.+s+[0‐9]+s+(.+)$/
spid = $1 # 1 () spid
spname = $2 # 2 () spname
evalue = $3 # 3 () evalue
if evalue =~ /^e/ # evalue e
evalue = “1.0” + evalue # 1.0
end
#

8. #
# evalue evalue_float
evalue_float = evalue.to_f
# spid_hash[spid] name spname evalue evalue_float
spid_hash[spid] = {:name=>spname, :evalue=>evalue_float}
end
end
# spid_hash k.step30.txt
# %s
out = open("k.step30.txt","w");
spid_hash.each {|spid,value|
out.prinv("%s|%s|%sn",spid,value[:name],value[:evalue]);
}
out.close
step20 blast k.step20.txt ID e‐value
k.step30.txt
‐ hsa:6469 ( ID kegg_id )
‐blast_clustalw.rb 28‐47 parse_blast
‐ last_clustalw.rb
b get_aaseq 84

9. : step30.rb
‐ if line =~ /^sp|(.+)|(.+)s+.+s+[0‐9]+s+(.+)$/
BLAST ID
sp
1 |
2 |
3
^: , |: |, .: , +: 1 , s: , $: ,
(): , []: []
Score E
Sequences producing significant alignments: (bits) Value
sp|Q15465|SHH_HUMAN Sonic hedgehog... 720 0.0
sp|Q62226|SHH_MOUSE Sonic hedgehog... 682 0.0
sp|Q63673|SHH_RAT Sonic hedgehog... 679 0.0
sp|Q91035|SHH_CHICK Sonic hedgehog... 605 e-172
sp|Q90385|SHH_CYNPY Sonic hedgehog... 510 e-144
spid
spname
evalue
spid_hash[spid][:name]
spid_hash[spid][:evalue]

10. (11):E‐value
(ex. 0.0, e‐100 )
query E‐value query
‐
*E‐value
*

11. (5) step40.rb
Blast E‐value
Step30 k.step30.txt E‐value
k.step40.txt
‐ hsa:6469 ( ID kegg_id )
‐blast_clustalw.rb 39‐43
‐blast_clustalw.rb e‐value 6
#!/usr/bin/env ruby
#
min_evalue = 1e‐170
# step30.rb
# spid_hash
lns_spid = File.read("k.step30.txt")
spid_hash = Hash.new
lns_spid.each_line do |line|
raise unless line =~ /^(.+)|(.+)|(.+)$/
spid = $1
spname = $2
evalue = $3
spid_hash[spid] =
{:name=>spname, :evalue=>evalue.to_f}
end #

12. #
#
#
spid_hash.each {|spid,value|
#
if value[:evalue] > min_evalue then
spid_hash.delete(spid) #
end
}
# k.step40.txt
out = open("k.step40.txt","w");
spid_hash.each {|spid,value|
out.prinv("%s|%s|%sn",
spid,value[:name],value[:evalue]);
}
out.close

13. (6) step50.rb
Sonic Hedgehog
‐ blast_clustalw.rb 49 59 87
#!/usr/bin/env ruby
require 'open‐uri’
#
# spid_hash
lns_spid = File.read("k.step40.txt")
spid_hash = Hash.new
lns_spid.each_line do |line|
raise unless line =~ /^(.+)|(.+)|(.+)$/
spid = $1
spname = $2
evalue = $3
spid_hash[spid] = {:name=>spname, :evalue=>evalue.to_f}
end
#

14. #
# spid_hash
spid_hash.each {|spid,value|
#
uri = "hap://togows.dbcls.jp/entry/ebi‐uniprot/" + spid + "/seq”
# REST
value[:seq] = open(uri).read
}
# fasta
out = open("k.step50.fa","w");
spid_hash.each {|spid,value|
out.prinv(">%s|%s|%sn",spid,value[:name],value[:evalue]);
out.prinv("%sn",value[:seq]);
}
out.close

15. (7) step60.rb
CLUSTALW alignment
Step50.rb clustalw SOAP
‐ blast_clustalw.rb 61 75 90
#!/usr/bin/env ruby
require 'soap/wsdlDriver’
# fasta query
query = File.read("k.step50.fa")
# query mulSple alignment
wsdl_clustalw = "hap://togows.dbcls.jp/soap/wsdl/ddbj_clustalw.wsdl"
serv_clustalw = SOAP::WSDLDriverFactory.new(wsdl_clustalw).create_rpc_driver
param = "‐align ‐matrix=blosum ‐gapdist=8 ‐maxdiv=40 ‐outorder=aligned ‐
pwmatrix=blosum”
# SOAP CLUSTALW
result_clustalw = serv_clustalw.DdbjClustalwAnalyzeParam(query,param)
# k.step60.txt
out = open("k.step60.txt","w");
out.puts result_clustalw
out.close

16. (8) blast_clustalw.rb
blast_clustalw.rb 77‐97 Step10‐60
shiy
# ID hsa:6469
# step10.rb
# step20.rb
#step30.rb step40.rb
#step50.rb
#step60.rb
# auto.txt

17. blast_clustalw.rb
1) hsa:6469 MIN_EVALUE ( 1.0e‐140, 1.0e‐120,
1.0e‐100 ) mulSple alignment ( )
2) rat Olr1082 mulSple alignment human mouse
ortholog
‐
rat Olr1082 UniProt ID
REST Entry retrieval UniProt KEGG ID
blast_clustalw.rb (MIN_EVALUE 1.0e‐100)
mulSple alignment ortholog
Ensembl Synteny ortholog

18. SOAP REST