Download 04_db_blast

Database Similarity Searching
BLAST
Global alignment of a pair of seqs., in which all residues from both seqs. are
included.
BLAST – local alignment
Interpreting BLAST output
-Smith and Waterman algorithm  guaranteed to find the best local alignment of
two seqs.
-Too slow in practice !!
-BLAST  heuristic search method that is not guaranteed to find the best local
alignment, but has been especially effective in practice
-e.g. S45649 (from a fossilized insect)
>gi|256517|gb|S45649.1| 16S rRNA [Mastotermes electrodominicus=termites,
amber-preserved fossil, Mitochondrial, 94 nt]
AATAAAATTTTAATAAATATAAAGATTTATAGGGTCTTCTCGGCCTTTAAAAATA
TTTTAGCCTTTTGAC AAAAAAAAAAAAATCTACAAAAAA
BLAST
http://www.ncbi.nlm.nih.gov/BLAST/
E-value, with the most significant hits listed first
E-value is the number of hits with the same level of
similarity that you would expect by chance
E = 0.01  occur once every 100 searches even when
there is no true match in the database
E-value is similar in spirit to the p-value of statistical
hypothesis tests.
P-value is the probability of finding a seq. similarity as
similar as the observed match if there were really no
true matches in the database.
E-value ≠ p-value
E-value ~ p-value when it is small (say < 0.1)
Since we are interested in unusual hits, it is safe to
interchange E-value with p-value.
E-value – the lower the better the alignment, matches
above 0.001 are often close to the twilight zone (not
significant)
Score (bits) – the higher the better the alignment, score
below 50 are unreliable
BLAST
The BLAST output may not be the same every time due to the upgrade of
several components :
Database, the BLAST program, the default parameters of the server
E-value, similarity and homology
Protein : >25 %, > 100 a.a., < 10-4
DNA : >70%, > 100 bp, < 10-4
Gap penalties
- constant penalty independent of the length of gap, A
- proportional penalty, penalty is proportional to the length L of the gap, BL
- Affine (『數』遠交的,『化學』親和的) gap penalty, gap-opening penalty +
gap-extension penalty = A+BL
Remark
• Prediction using similarity is a powerful idea in bioinformatics
• homologue  seqs. evolved by divergence from a common ancestor,
therefore to say two seqs. share 50% homology is nonsense; to say two
seqs. share 50% similarity and that they indicate possible homology is
the correct usage of the terms
• Similarity NOT necessary implied homology
BLAST (choosing the parameters)
BLAST - Most highly cited paper >12000 times
alternative methods  seeds + dynamics programming  speed up, faster
not guaranteed to find the best alignment  less accurate
BLAST (Sequence filters)
http://www.ncbi.nlm.nih.gov/BLAST/
BLAST
What is a coiled-coil?
Coiled-coil domains are characterized by a heptad
(成七的一組) repeat pattern in which residues in
the first and fourth position are hydrophobic, and
residues in the fifth and seventh position are
predominantly charged or polar. This pattern can
be used by computational methods, such as
MultiCoil (MIT) or SOCKET (University of
Sussex)to predict coiled-coil domains in amino
acid sequences.
BLAST programs
BLASTing DNA sequences
Use of BLASTx to find ORF
AE008569
Use of BLASTx to find ORF
Frame = +1
Frame = -2
Use of BLASTx to find ORF
Use of BLASTx to find ORF
Use of BLASTx to find ORF
BLAST procedures
BLAST
 ls
• The E-value of the BLAST is given by E  kmne
• where k (depend on the scoring matrix and gap penalty combination) and l
are constants, m and n denote the seqs. length, s is the scaling factor for the
scoring matrix used
• Gumbel extreme value distribution for alignment scores
 kmne lx
•
P  1 e
http://www.itl.nist.gov/div898/handbook/eda/section3/eda366g.htm
Position-Specific Iterated BLAST (PSI-BLAST)
Position-Specific Iterated BLAST (PSI-BLAST)
Query sequence – human hemoglobin
>gi|57013850|sp|P69905|HBA_HUMAN Hemoglobin alpha subunit (Hemoglobin alpha chain) (Alpha-globin)
MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPN
ALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSK YR
0 ≦E-value < 10-40
Position-Specific Iterated BLAST (PSI-BLAST)
Query sequence – human hemoglobin
>gi|57013850|sp|P69905|HBA_HUMAN Hemoglobin alpha subunit (Hemoglobin alpha chain) (Alpha-globin)
MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHA
HKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSK YR
Gene or
Structure
information
Position-Specific Iterated BLAST (PSI-BLAST)
More seqs. are identified than
Iteration 1
Position-Specific Iterated BLAST (PSI-BLAST)
Add or remove the hits that seems
to be relevant or irrelevant (non-human seq.)
Position-Specific Iterated BLAST (PSI-BLAST)
B~C