• Navigation
  • Get Started
  • News
  • FAQs
  • Tutorials
  • Documentation
  • Glossary
  • References
  • System Support
  • Contact Us
  • Recent changes
• CoGe links
  • CoGe Home
  • OrganismView
  • FeatViewer
  • CoGeBlast
  • SynMap
  • GEvo
• Sites Linked to CoGe
  • PlantGDB
  • MaizeGDB
• Sites Linked from CoGe
  • ViennaFold
  • TARGeT
  • Phylogeny.fr
  • Prosite
• Toolbox
  • What links here
  • Related changes
  • Special pages
  • Printable version
  • Permanent link

GEvo Blastn Bug

From CoGepedia

Contents 1 Bug Description 2 Visualization 3 Report of "disappearing" HSP in blast file 4 Blast Commands 5 Conclusions 6 Download 7 Comparison to Blast 2.2.25+

Bug Description

When using blastn in GEvo, a large HSP appear in the middle of the region when the query sequence length is changed by 1 nucleotide.

This problem was identified by Mike Freeling.

Visualization

Difference between analyses is that addition of 1 nucleotide to the top panel of the top analysis

Report of "disappearing" HSP in blast file

Top portion of blast report which contains the large HSP (problem HSP starts at "Query 129"):

BLASTN 2.2.24+

Query=  AT1G75520 
Length=478

Subject=  Bra008203 
Length=17361


 Score = 68.0 bits (35),  Expect = 3e-14
 Identities = 76/94 (80%), Gaps = 4/94 (4%)
 Strand=Plus/Plus

Query  1      CTAGGTTTCGTGTTCCACTGATCAAAGATTTGAAAAAAAACATATACTTAGTAAACTTCA  60
              |||||||| ||||||||||||||||||| ||    || |||||||    |  |||||| |
Sbjct  9943   CTAGGTTTTGTGTTCCACTGATCAAAGAGTT----AAGAACATATTTCAATAAAACTTTA  9998

Query  61     AGCAATTTTTATATTACCCAATTGAATTTCTCCA  94
              |  |||||||||| |||||| |||||||||||||
Sbjct  9999   AATAATTTTTATACTACCCAGTTGAATTTCTCCA  10032


 Score = 54.5 bits (28),  Expect = 3e-10
 Identities = 32/34 (94%), Gaps = 0/34 (0%)
 Strand=Plus/Plus

Query  442    TTGGCGTAGATGAATGTAAACGGATGGTAATATA  475
              |||||||| |||||||||||||||| ||||||||
Sbjct  10342  TTGGCGTATATGAATGTAAACGGATAGTAATATA  10375


 Score = 50.7 bits (26),  Expect = 4e-09
 Identities = 77/95 (81%), Gaps = 5/95 (5%)
 Strand=Plus/Plus

Query  129    ATATATATATACCCAACAACTGAGAAAAGATGGAAAAAGTTTAGTTAAAAACTGGTCCTG  188
              ||||||||||| |||||| ||  |||||||||||| || || |||||||||     | | 
Sbjct  10072  ATATATATATAACCAACATCTCGGAAAAGATGGAACAAATT-AGTTAAAAAAA---CATT  10127

Query  189    GGCGGCTTTAAATTATATTTATGCACTTAAATTTA  223
              ||||||||||||| ||||| ||| | |||||||||
Sbjct  10128  GGCGGCTTTAAATCATATTCATGTA-TTAAATTTA  10161


 Score = 48.8 bits (25),  Expect = 2e-08
 Identities = 47/58 (81%), Gaps = 0/58 (0%)
 Strand=Plus/Plus

Query  316    GGTTTTTACTTAGATAATATCGTGTCATTCCATCTAGATTCAACCCCTGTCTACAATA  373
              |||||| ||| |||| |||| ||  || | |  ||||||||||||||| |||||||||
Sbjct  10207  GGTTTTCACTGAGATGATATTGTTCCAGTTCCACTAGATTCAACCCCTCTCTACAATA  10264


 Score = 25.7 bits (13),  Expect = 0.15
 Identities = 13/13 (100%), Gaps = 0/13 (0%)
 Strand=Plus/Plus

Query  127   TAATATATATATA  139
             |||||||||||||
Sbjct  7327  TAATATATATATA  7339

Top portion of blast report which DOES NOT contains the large HSP :

BLASTN 2.2.24+


Query=  AT1G75520 
Length=479

Subject=  Bra008203 
Length=17361


 Score = 69.9 bits (36),  Expect = 7e-15
 Identities = 77/95 (81%), Gaps = 4/95 (4%)
 Strand=Plus/Plus

Query  1      ACTAGGTTTCGTGTTCCACTGATCAAAGATTTGAAAAAAAACATATACTTAGTAAACTTC  60
              ||||||||| ||||||||||||||||||| ||    || |||||||    |  |||||| 
Sbjct  9942   ACTAGGTTTTGTGTTCCACTGATCAAAGAGTT----AAGAACATATTTCAATAAAACTTT  9997

Query  61     AAGCAATTTTTATATTACCCAATTGAATTTCTCCA  95
              ||  |||||||||| |||||| |||||||||||||
Sbjct  9998   AAATAATTTTTATACTACCCAGTTGAATTTCTCCA  10032


 Score = 54.5 bits (28),  Expect = 3e-10
 Identities = 32/34 (94%), Gaps = 0/34 (0%)
 Strand=Plus/Plus

Query  443    TTGGCGTAGATGAATGTAAACGGATGGTAATATA  476
              |||||||| |||||||||||||||| ||||||||
Sbjct  10342  TTGGCGTATATGAATGTAAACGGATAGTAATATA  10375


 Score = 48.8 bits (25),  Expect = 2e-08
 Identities = 47/58 (81%), Gaps = 0/58 (0%)
 Strand=Plus/Plus

Query  317    GGTTTTTACTTAGATAATATCGTGTCATTCCATCTAGATTCAACCCCTGTCTACAATA  374
              |||||| ||| |||| |||| ||  || | |  ||||||||||||||| |||||||||
Sbjct  10207  GGTTTTCACTGAGATGATATTGTTCCAGTTCCACTAGATTCAACCCCTCTCTACAATA  10264


 Score = 25.7 bits (13),  Expect = 0.15
 Identities = 13/13 (100%), Gaps = 0/13 (0%)
 Strand=Plus/Plus

Query  128   TAATATATATATA  140
             |||||||||||||
Sbjct  7327  TAATATATATATA  7339

Top portion of blast report which contains the large HSP :br>

Lambda     K      H
    1.33    0.621     1.12 

Gapped
Lambda     K      H
    1.33    0.621     1.12 

Effective search space used: 8066820




Matrix: blastn matrix 1 -2
Gap Penalties: Existence: 5, Extension: 2

Top portion of blast report which DOES NOT contains the large HSP :

Lambda     K      H
    1.33    0.621     1.12 

Gapped
Lambda     K      H
    1.33    0.621     1.12 

Effective search space used: 8084168




Matrix: blastn matrix 1 -2
Gap Penalties: Existence: 5, Extension: 2

Blast Commands

Extra HSP, Legacy Blast:

/usr/local/bin/legacy_blast.pl bl2seq -p blastn -o /opt/apache/CoGe/tmp/GEvo/52376346_1-2.bl2seq -i /opt/apache/CoGe/tmp/GEvo/f34524790ds39598r1c1u-2233d777g2dsg3.faa -j /opt/apache/CoGe/tmp/GEvo/f103007203ds48732r1cA02u8000d8000g1dsg12468.faa  -W 7 -G 5 -E 2 -q -2 -r 1 -e 30 -F F

Extra HSP, Blast+

blastn -query /opt/apache/CoGe/tmp/GEvo/f34524790ds39598r1c1u-2233d777g2dsg3.faa -subject /opt/apache/CoGe/tmp/GEvo/f103007203ds48732r1cA02u8000d8000g1dsg12468.faa -evalue 30 -gapopen 5 -gapextend 2 -word_size 7 -penalty -2 -reward 1 -dust no

Missing HSP, Legacy Blast

/usr/local/bin/legacy_blast.pl bl2seq -p blastn -o /opt/apache/CoGe/tmp/GEvo/22224470_1-2.bl2seq -i /opt/apache/CoGe/tmp/GEvo/f34524790ds39598r1c1u-2232d777g2dsg3.faa -j /opt/apache/CoGe/tmp/GEvo/f103007203ds48732r1cA02u8000d8000g1dsg12468.faa  -W 7 -G 5 -E 2 -q -2 -r 1 -e 30 -F F

Missing HSP, Blast+

blastn -query /opt/apache/CoGe/tmp/GEvo/f34524790ds39598r1c1u-2232d777g2dsg3.faa -subject /opt/apache/CoGe/tmp/GEvo/f103007203ds48732r1cA02u8000d8000g1dsg12468.faa -evalue 30 -gapopen 5 -gapextend 2 -word_size 7 -penalty -2 -reward 1 -dust no

Conclusions

• The bug is in blastn. Current version is 2.2.24
• Possible causes:
  • Change in sequence search space (sequence length) causes change in evalue
  • Change in sequence causes an "edge-effect" where exact sequence pattern at the end of the character sequence causes a chance in HSP identification
  • Repeat sequences are causing an internal problem to how blast identifies and categorizes HSPs

Download

The sequences, blast reports, log files, and GEvo Images can be obtained at: http://genomevolution.org/CoGe/data/distrib/gevo_blast_bug.tar.gz

Comparison to Blast 2.2.25+

Same problem exists: GEvo bug Blast 2.2.25+