[Bioperl-l] different results with remote-blast skript
Smithies, Russell
Russell.Smithies at agresearch.co.nz
Mon Jul 6 16:56:41 EDT 2009
Hi Jonas,
You can't just play with the BLAST parameters and hope for a "better" result.
I'd suggest that if you aren't sure what they do, you should leave them alone as small changes can make huge differences in the output - it's quite possible to miss finding what you're looking for by using the wrong parameters.
If all else fails, read the blast manual: http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/blastall/blastall_all.html
http://www.ncbi.nlm.nih.gov/blast/tutorial/
Or Read Ian Korfs' excellent book: http://books.google.com/books?id=xvcnhDG9fNUC&lpg=PR17&ots=WJpfuHF6Hn&dq=ian%20korf%20%20blast%20book&pg=PA3
Don't worry about the integer overflow bug as there's nothing you can do about it. If you're interested, Google and Wikipedia are your friends: http://en.wikipedia.org/wiki/Integer_overflow
Russell
> -----Original Message-----
> From: bioperl-l-bounces at lists.open-bio.org [mailto:bioperl-l-
> bounces at lists.open-bio.org] On Behalf Of Jonas Schaer
> Sent: Tuesday, 7 July 2009 12:14 a.m.
> To: BioPerl List; Chris Fields
> Subject: Re: [Bioperl-l] different results with remote-blast skript
>
> Hi guys, thanks for your answers so far.
> @jason: integer overflow in blast.... sorry, but what do you mean by that?
> how can I fix it...?
>
> Since I never really changed any parameters I thought them all to be default.
> whatever, I tried to get "better" results with my prog by changing
> these:
> $Bio::Tools::Run::RemoteBlast::HEADER{'GAPCOSTS'} = '11 1';
> $Bio::Tools::Run::RemoteBlast::HEADER{'MAX_NUM_SEQ'} = '100';
> $Bio::Tools::Run::RemoteBlast::HEADER{'EXPECT'} = '10';
> $Bio::Tools::Run::RemoteBlast::HEADER{'COMPOSITION_BASED_STATISTICS'} = '1';
> with no effect...I guess these were default values anyway.
>
> So please maybe you can tell me all the other parameters I can change with my
> perl-skript AND how to do that?
> Unfortunately both, perl and the blast-algorithm are pretty much new to me,
> maybe thats why I just cannot find out how to do that on my own... :/
>
> Here is the output I get with my remote-blast skript:
> ##############################################################################
> ###################################
> Query Name:
> MGSSSVGTYHLLLVLMGAGGEQQAVQAGAEVASTEQVDGSGMAANSRGSTSGSEQPPRDSDLGLLRSL
> L
> hit name is ref|XP_001702807.1|
> score is 442
> BLASTP 2.2.21+
> Reference: Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer,
> Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped
> BLAST and PSI-BLAST: a new generation of protein database search programs",
> Nucleic Acids Res. 25:3389-3402.
>
>
> Reference for composition-based statistics: Alejandro A.
> Schaffer, L. Aravind, Thomas L. Madden, Sergei Shavirin, John L. Spouge, Yuri
> I. Wolf, Eugene V. Koonin, and Stephen F. Altschul (2001), "Improving the
> accuracy of PSI-BLAST protein database searches with composition-based
> statistics and other refinements", Nucleic Acids Res. 29:2994-3005.
>
>
> RID: 53STX5G2013
>
>
> Database: All non-redundant GenBank CDS
> translations+PDB+SwissProt+PIR+PRF excluding environmental samples
> from WGS projects
> 9,252,587 sequences; 3,169,972,781 total letters Query=
> MGSSSVGTYHLLLVLMGAGGEQQAVQAGAEVASTEQVDGSGMAANSRGSTSGSEQPPRDSDLGLLRSLL
> DVAGVDRTALEVKLLALAEAGAEMPPAQDSQATAAGVVATLTSVYRQQVARAWHERDDNAFRQAHQNTAM
> ATGPDPDDEYE
> Length=150
>
>
> Score
> E
> Sequences producing significant alignments: (Bits)
> Value
>
> ref|XP_001702807.1| ClpS-like protein [Chlamydomonas reinhard... 174
> 2e-42
>
>
> ALIGNMENTS
> >ref|XP_001702807.1| ClpS-like protein [Chlamydomonas reinhardtii]
> gb|EDP06586.1| ClpS-like protein [Chlamydomonas reinhardtii]
> Length=303
>
> Score = 174 bits (442), Expect = 2e-42, Method: Composition-based stats.
> Identities = 150/150 (100%), Positives = 150/150 (100%), Gaps = 0/150 (0%)
>
> Query 1 MGSSSVGTYHLLLVLMgaggeqqavqagaevaSTEQVDGSGMAANSRGSTSGSEQPPrds 60
> MGSSSVGTYHLLLVLMGAGGEQQAVQAGAEVASTEQVDGSGMAANSRGSTSGSEQPPRDS
> Sbjct 154 MGSSSVGTYHLLLVLMGAGGEQQAVQAGAEVASTEQVDGSGMAANSRGSTSGSEQPPRDS
> 213
>
> Query 61 dlgllrslldVAGVDRTalevkllalaeagaeMPPAQDSQATAAGVVATLTSVYRQQVAR
> 120
> DLGLLRSLLDVAGVDRTALEVKLLALAEAGAEMPPAQDSQATAAGVVATLTSVYRQQVAR
> Sbjct 214 DLGLLRSLLDVAGVDRTALEVKLLALAEAGAEMPPAQDSQATAAGVVATLTSVYRQQVAR
> 273
>
> Query 121 AWHERDDNAFRQAHQNTAMATGPDPDDEYE 150
> AWHERDDNAFRQAHQNTAMATGPDPDDEYE
> Sbjct 274 AWHERDDNAFRQAHQNTAMATGPDPDDEYE 303
>
>
>
> Database: All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF
> excluding environmental samples from WGS projects
> Posted date: Jul 5, 2009 4:41 AM
> Number of letters in database: -1,124,994,511
> Number of sequences in database: 9,252,587
>
> Lambda K H
> 0.309 0.122 0.345
> Gapped
> Lambda K H
> 0.267 0.0410 0.140
> Matrix: BLOSUM62
> Gap Penalties: Existence: 11, Extension: 1
> Number of Sequences: 9252587
> Number of Hits to DB: 60273703
> Number of extensions: 1448367
> Number of successful extensions: 2103
> Number of sequences better than 10: 0
> Number of HSP's better than 10 without gapping: 0
> Number of HSP's gapped: 2113
> Number of HSP's successfully gapped: 0
> Length of query: 150
> Length of database: 3169972781
> Length adjustment: 113
> Effective length of query: 37
> Effective length of database: 2124430450
> Effective search space: 78603926650
> Effective search space used: 78603926650
> T: 11
> A: 40
> X1: 16 (7.1 bits)
> X2: 38 (14.6 bits)
> X3: 64 (24.7 bits)
> S1: 42 (20.8 bits)
> S2: 74 (33.1 bits)
>
> ##############################################################################
> ###################################
> and here are the hits (?) of the blast-algorithm on the ncbi-homepage with
> the same query of course:
> ref|XP_001702807.1| ClpS-like protein [Chlamydomonas reinhard... 300
> 3e-80
> ref|XP_001942719.1| PREDICTED: similar to GA16705-PA [Acyrtho... 36.2
> 1.1
> ref|ZP_03781446.1| hypothetical protein RUMHYD_00880 [Blautia... 35.4
> 1.8
> ref|XP_001563232.1| leucyl-tRNA synthetase [Leishmania brazil... 34.3
> 4.2
> ref|XP_680841.1| hypothetical protein AN7572.2 [Aspergillus n... 33.5
> 6.0
> ref|YP_001768110.1| hypothetical protein M446_1150 [Methyloba... 33.5
> 7.0
> ##############################################################################
> ###################################at
> least the first hit is the same, but even there there is a different score
> and e-value.
>
> thanks so much for any help :)
> regards, jonas
>
>
> ----- Original Message -----
> From: "Chris Fields" <cjfields at illinois.edu>
> To: "Jason Stajich" <jason at bioperl.org>
> Cc: "Smithies, Russell" <Russell.Smithies at agresearch.co.nz>; "'BioPerl
> List'" <bioperl-l at lists.open-bio.org>; "'Jonas Schaer'"
> <Jonas_Schaer at gmx.de>
> Sent: Monday, July 06, 2009 12:51 AM
> Subject: Re: [Bioperl-l] different results with remote-blast skript
>
>
> > That inspires confidence ;>
> >
> > chris
> >
> > On Jul 5, 2009, at 4:40 PM, Jason Stajich wrote:
> >
> >> integer overflow in blast....
> >>
> >> On Jul 5, 2009, at 2:00 PM, Smithies, Russell wrote:
> >>
> >>> I'd guess it's a difference in the parameters used.
> >>> Interesting that both have the number of letters in the db as
> >>> "-1,125,070,205", I assume that's a bug :-)
> >>>
> >>> Stats from your remote_blast:
> >>>
> >>> 'stats' => {
> >>> 'S1' => '42',
> >>> 'S1_bits' => '20.8',
> >>> 'lambda' => '0.309',
> >>> 'entropy' => '0.345',
> >>> 'kappa_gapped' => '0.0410',
> >>> 'T' => '11',
> >>> 'kappa' => '0.122',
> >>> 'X3_bits' => '24.7',
> >>> 'X1' => '16',
> >>> 'lambda_gapped' => '0.267',
> >>> 'X2' => '38',
> >>> 'S2' => '74',
> >>> 'seqs_better_than_cutoff' => '0',
> >>> 'posted_date' => 'Jul 4, 2009 4:41 AM',
> >>> 'Hits_to_DB' => '60102303',
> >>> 'dbletters' => '-1125070205',
> >>> 'A' => '40',
> >>> 'num_successful_extensions' => '2004',
> >>> 'num_extensions' => '1436892',
> >>> 'X1_bits' => '7.1',
> >>> 'X3' => '64',
> >>> 'entropy_gapped' => '0.140',
> >>> 'dbentries' => '9252258',
> >>> 'X2_bits' => '14.6',
> >>> 'S2_bits' => '33.1'
> >>> }
> >>>
> >>>
> >>> Stats from a blast done on the NCBI webpage:
> >>>
> >>> Database: All non-redundant GenBank CDS translations+PDB+SwissProt
> >>> +PIR+PRF
> >>> excluding environmental samples from WGS projects
> >>> Posted date: Jul 4, 2009 4:41 AM
> >>> Number of letters in database: -1,125,070,205
> >>> Number of sequences in database: 9,252,258
> >>>
> >>> Lambda K H
> >>> 0.309 0.124 0.340
> >>> Gapped
> >>> Lambda K H
> >>> 0.267 0.0410 0.140
> >>> Matrix: BLOSUM62
> >>> Gap Penalties: Existence: 11, Extension: 1
> >>> Number of Sequences: 9252258
> >>> Number of Hits to DB: 86493230
> >>> Number of extensions: 3101413
> >>> Number of successful extensions: 9001
> >>> Number of sequences better than 100: 65
> >>> Number of HSP's better than 100 without gapping: 0
> >>> Number of HSP's gapped: 9000
> >>> Number of HSP's successfully gapped: 66
> >>> Length of query: 150
> >>> Length of database: 3169897087
> >>> Length adjustment: 113
> >>> Effective length of query: 37
> >>> Effective length of database: 2124391933
> >>> Effective search space: 78602501521
> >>> Effective search space used: 78602501521
> >>> T: 11
> >>> A: 40
> >>> X1: 16 (7.1 bits)
> >>> X2: 38 (14.6 bits)
> >>> X3: 64 (24.7 bits)
> >>> S1: 42 (20.8 bits)
> >>> S2: 65 (29.6 bits)
> >>>
> >>>
> >>>> -----Original Message-----
> >>>> From: bioperl-l-bounces at lists.open-bio.org [mailto:bioperl-l-
> >>>> bounces at lists.open-bio.org] On Behalf Of Jonas Schaer
> >>>> Sent: Sunday, 28 June 2009 10:15 p.m.
> >>>> To: BioPerl List
> >>>> Subject: [Bioperl-l] different results with remote-blast skript
> >>>>
> >>>> Hi again :)
> >>>> please, I only have this little question:
> >>>> why do I get different results with my remote::blast perl skript
> >>>> then on the
> >>>> ncbi blast homepage?
> >>>> I am using blastp, the query is an amino-sequence (different
> >>>> results with any
> >>>> sequence, differences not only in number of hits but even in e-
> >>>> values, scores
> >>>> etc...), the database is 'nr'.
> >>>> PLEASE help me,
> >>>> thank you in advance,
> >>>> Jonas
> >>>>
> >>>> ps: my skript:
> >>>>
> ##############################################################################
> >>>> ##
> >>>> use Bio::Seq::SeqFactory;
> >>>> use Bio::Tools::Run::RemoteBlast;
> >>>> use strict;
> >>>> my @blast_report;
> >>>> my $prog = 'blastp';
> >>>> my $db = 'nr';
> >>>> my $e_val= '1e-10';
> >>>> #my $e_val= '10';
> >>>> my @params = ( '-prog' => $prog,
> >>>> '-data' => $db,
> >>>> '-expect' => $e_val,
> >>>> '-readmethod' => 'SearchIO' );
> >>>> my $factory = Bio::Tools::Run::RemoteBlast->new(@params);
> >>>> $Bio::Tools::Run::RemoteBlast::HEADER{'GAPCOSTS'} = '11 1';
> >>>> $Bio::Tools::Run::RemoteBlast::HEADER{'MAX_NUM_SEQ'} = '100';
> >>>> $Bio::Tools::Run::RemoteBlast::HEADER{'EXPECT'} = '10';
> >>>> $
> >>>> Bio
> >>>> ::Tools::Run::RemoteBlast::HEADER{'COMPOSITION_BASED_STATISTICS'}
> >>>> = '1';
> >>>>
> >>>> my
> >>>> $
> >>>> blast_seq
> >>>> ='MGSSSVGTYHLLLVLMGAGGEQQAVQAGAEVASTEQVDGSGMAANSRGSTSGSEQPPRDSDLGLLR
> >>>>
> SLLDVAGVDRTALEVKLLALAEAGAEMPPAQDSQATAAGVVATLTSVYRQQVARAWHERDDNAFRQAHQNTAMATGPD
> >>>> PDDEYE';
> >>>> #$v is just to turn on and off the messages
> >>>> my $v = 1;
> >>>> my $seqbuilder = Bio::Seq::SeqFactory->new('-type' =>
> >>>> 'Bio::PrimarySeq');
> >>>> my $seq = $seqbuilder->create(-seq =>$blast_seq, -display_id =>
> >>>> "$blast_seq");
> >>>> my $filename='temp2.out';
> >>>> my $r = $factory->submit_blast($seq);
> >>>> print STDERR "waiting..." if( $v > 0 );
> >>>> while ( my @rids = $factory->each_rid )
> >>>> {
> >>>> foreach my $rid ( @rids )
> >>>> {
> >>>> my $rc = $factory->retrieve_blast($rid);
> >>>> if( !ref($rc) )
> >>>> {
> >>>> if( $rc < 0 )
> >>>> {
> >>>> $factory->remove_rid($rid);
> >>>> }
> >>>> print STDERR "." if ( $v > 0 );
> >>>> }
> >>>> else
> >>>> {
> >>>> my $result = $rc->next_result();
> >>>> $factory->save_output($filename);
> >>>> $factory->remove_rid($rid);
> >>>> print "\nQuery Name: ", $result->query_name(),
> >>>> "\n";
> >>>> while ( my $hit = $result->next_hit )
> >>>> {
> >>>> next unless ( $v > 0);
> >>>> print "\thit name is ", $hit->name, "\n";
> >>>> while( my $hsp = $hit->next_hsp )
> >>>> {
> >>>> print "\t\tscore is ", $hsp->score, "\n";
> >>>> }
> >>>> }
> >>>> }
> >>>> }
> >>>>
> >>>>
> >>>> }
> >>>> @blast_report = get_file_data ($filename);
> >>>> return @blast_report;
> >>>>
> ##############################################################################
> >>>> ####
> >>>> _______________________________________________
> >>>> Bioperl-l mailing list
> >>>> Bioperl-l at lists.open-bio.org
> >>>> http://lists.open-bio.org/mailman/listinfo/bioperl-l
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> >> --
> >> Jason Stajich
> >> jason at bioperl.org
> >>
> >> _______________________________________________
> >> Bioperl-l mailing list
> >> Bioperl-l at lists.open-bio.org
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