BLAST and Multiple Sequence Alignment Learning objectives-Learn the basics of BLAST, Psi-BLAST, and multiple sequence alignment
Dec 21, 2015
BLAST and Multiple Sequence Alignment
Learning objectives-Learn the basics of BLAST, Psi-BLAST, and multiple sequence alignment
Which program should one use?
Most researchers use methods for determining local similarities: Smith-Waterman (gold standard) FASTA BLAST }Do not find every possible alignment
of query with database sequence. Theseare used because they run faster than S-W
BLAST
Basic Local Alignment Search Tool
Three phases:
1) List of high scoring words
2) Scan the sequence database
3) Extend hits
The threshold and word size
The program declares a hit if the word taken from the query sequence has a score >= T when a scoring matrix is used.
This allows the word size (W) to be kept high (for speed) without sacrificing sensitivity.
If T is increased, the number of background hits is reduced and the program will run faster.
Phase 1: Compile a list of high-scoring words above threshold T.Query sequence: human p53: . . . RCPHHERCSD. . .Words derived from query sequence: RCP, CPH, PHH, HHE, …List of words above threshold T:
Word Scores from BLOSUM scoring matrix
Total score
RCP 5 + 9 + 7 21
KCP 2 + 9 + 7 18
QCP 1 + 9 + 7 17
ECP 0 + 9 + 7 16
Note: The line is located at the threshold.Word size is 3.
. . .
. . .
Phase 2: Scan the database for short segments that match the list of acceptable words/scores above or equal to threshold T.
Phase 3: Extend the hits and terminate when the tabulated score drops below a cutoff score.
Query EVVRRCPHHERCSD EVVRRCPHHER S+Sbjct EVVRRCPHHERSSE (Ch. hamster p53 O09185)
If the hit is extended far enough the query/subj segmentis called a High Scoring Segment Pair (HSP).
What are the different BLAST programs?
blastp compares an amino acid query sequence against a protein sequence
database blastn compares a nucleotide query sequence against a nucleotide sequence
database blastx compares a nucleotide query sequence translated in all reading frames
against a protein sequence database tblastn compares a protein query sequence against a nucleotide sequence database
dynamically translated in all reading frames tblastx compares the six-frame translations of a nucleotide query sequence against
the six-frame translations of a nucleotide sequence database. Please note that tblastx program cannot be used with the nr database on the BLAST Web page.
What are the different BLAST programs? (continued)
psi-blast Compares a protein sequence to a protein database. Performs the
comparison in an iterative fashion in order to detect homologs that are evolutionarily distant.
blast2 Compares two protein or two nucleotide sequences.
The E value (false positive expectation value)
The Expect value (E) is a parameter that describes the number of “hits” one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially as the Similarity Score (S) increases (inverse relationship). The higher the Similarity Score, the lower the E value. Essentially, the E value describes the random background noise that exists for matches between two sequences. The E value is used as a convenient way to create a significance threshold for reporting results. When the E value is increased from the default value of 10 prior to a sequence search, a larger list with more low-similarity scoring hits can be reported. An E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size you might expect to see 1 match with a similar score simply by chance.
E value (Karlin-Altschul statistics)E = K•m•n•e-λS
Where K is a scaling factor (constant), m is the length of the query sequence, n is the length of the database sequence, λ is the decay constant, S is the similarity score.
If S increases, E decreases exponentially.If the decay constant increases, E decreases exponentiallyIf m•n increases the “search space” increases and there is a greater
chance for a random “hit”, E increases. Larger database will increase E. However, larger query sequence often decreases E. Why???
Thought problem
A homolog to a query sequence resides in two databases. One is the UniProtKB/SwissProt database and the other is the PDB database. After performing BLAST search against the UniProtKB database you obtain an E value of 1. After performing the BLAST search against the PDB database you obtain an E value of 0.0625. What is the relative sizes of the two databases?
Using BLAST to get quick answers to bioinformatics problems
Task BLAST method Trad. Method
Predict protein function (1)
Perform blastp on PIR or Swiss-Prot database
Perform wet-lab experiment
Predict protein function (2)
Perform tblastn on NR database
Perform wet-lab experiment
Predict protein structure
Perform blastp against PDB
Structure prediction software, x-ray crystall., NMR
Using BLAST to get quick answers to bioinformatics problems (cont.)
Task BLAST method Trad. Method
Locate genes in a genome
Divide genome into 2-5 kb sequences. Perform blastx against NR protein datbase
Run gene prediction software. Perform microarray analysis or RNAs
Find distantly related proteins
Perform psi-blast No traditional method
Identify DNA sequence
Perform blastn Screen genomic DNA library
Filtering Repetitive Sequences
Over 50% of genomic DNA is repetitiveThis is due to: retrotransposons ALU region microsatellites centromeric sequences, telomeric sequences 5’ Untranslated Region of ESTs
Example of EST with simple low complexity region:
T27311GGGTGCAGGAATTCGGCACGAGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC
PSI-BLAST
PSI-position specific iterativea position specific scoring matrix (PSSM) is constructed automatically from multiple HSPs of initial BLAST search. Normal E value threshold is used.The PSSM is created as the new scoring matrix for a second BLAST search. A low E value threshold is used (E=.001).Result-1) obtains distantly related sequences
2) finds the important residues that provide function or structure.
Steps to multiple alignment
Create Alignment
Edit the alignment to ensure that regions of functionalor structural similarity are preserved
PhylogeneticAnalysis
StructureAnalysis
Find conserved motifsto deduce function
Design ofPCR primers
Multiple Sequence Alignment
Collection of three or more protein (or nucleic acid) sequences partially or completely aligned.
Aligned residues tend to occupy corresponding positions in the 3-D structure of each aligned protein.
Practical use of MSA
Helps to place protein into a group of related proteins. It will provide insight into function, structure and evolution.
Helps to detect homologs
Identifies sequencing errors
Identifies important regulatory regions in the promoters of genes.
Clustal W (Thompson et al., 1994)
CLUSTAL=Cluster alignment
The underlying concept is that groups of sequences are phylogenetically related. If they can be aligned then one can construct a phylogenetic tree.
Flowchart of computation steps in Clustal W (Thompson et al., 1994)
Pairwise alignment: calculation of distance matrix
Creation of unrooted neighbor-joining tree
Rooted nJ tree (guide tree) and calculation of sequence weights
Progressive alignment following the guide tree
Step 1-Pairwise alignments
Compare each sequence with eachother and calculate a distance matrix.
A -
B .87 -
C .59 .60 -
A B C
Each number represents the numberof exact matches divided by thesequence length (ignoring gaps).Thus, the higher the number the moreclosely related the two sequences are.
In this matrix, sequence A is 87% identical to sequence B
Different sequences
Step 1-Pairwise alignments
Compare each sequence with eachother and pairwise alignment scores
human EYSGSSEKIDLLASDPHEALICKSERVHSKSVESNIEDKIFGKTYRKKASLPNLSHVTEN 480dog EYSGSSEKIDLMASDPQDAFICESERVHTKPVGGNIEDKIFGKTYRRKASLPKVSHTTEV 477mouse GGFSSSRKTDLVTPDPHHTLMCKSGRDFSKPVEDNISDKIFGKSYQRKGSRPHLNHVTE 476
Step 1-Calculation of Distance Matrix
Use the Distance Matrix to create a Guide Tree todetermine the “order” of the sequences.
I =D = 1 – (I) D = Difference score
# of identical aa’s in pairwise global alignmenttotal number of aa’s in shortest sequence
Hbb-Hu 1 -
Hbb-Ho 2 .17 -
Hba-Hu 3 .59 .60 -
Hba-Ho 4 .59 .59 .13 -
Myg-Ph 5 .77 .77 .75 .75 -
Gib-Pe 6 .81 .82 .73 .74 .80 -
Lgb-Lu 7 .87 .86 .86 .88 .93 .90 -
1 2 3 4 5 6 7
Step 3-Create Rooted NJ Tree
Weight
AlignmentOrder of alignment:1 Hba-Hu vs Hba-Ho2 Hbb-Hu vs Hbb-Ho3 A vs B4 Myg-Ph vs C5 Gib-Pe vs D6 Lgh-Lu vs E
Rules for alignment
Short stretches of 5 hydrophilic residues often indicate loop or random coil regions (not essential for structure) and therefore gap penalties are reduced reduced for such stretches.Gap penalties for closely related sequences are lowered compared to more distantly related sequences (“once a gap always a gap” rule). It is thought that those gaps occur in regions that do not disrupt the structure or function.Alignments of proteins of known structure show that proteins gaps do not occur more frequently than every eight residues. Therefore penalties for gaps increase when required at 8 residues or less for alignment. This gives a lower alignment score in that region.A gap weight is assigned after each aa according the frequency that such a gap naturally occurs after that aa in nature
Amino acid weight matrices
As we know, there are many scoring matrices that one can use depending on the relatedness of the aligned proteins.As the alignment proceeds to longer branches the aa scoring matrices are changed to more divergent scoring matrices. The length of the branch is used to determine which matrix to use and contributes to the alignment score.
Example of Sequence Alignment using Clustal W
Asterisk represents identity: represents high similarity. represents low similarity
Multiple Alignment Considerations
Quality of guide tree. It would be good to have a set of closely related sequences in the alignment to set the pattern for more divergent sequences.If the initial alignments have a problem, the problem is magnified in subsequent steps.CLUSTAL W is best when aligning sequences that are related to each other over their entire lengthsDo not use when there are variable N- and C- terminal regionsIf protein is enriched for G,P,S,N,Q,E,K,R then these residues should be removed from gap penalty list. (what types of residues are these?)
Reference: http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/