BLAST MCDB 187. Friday, February 8, 13
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1 BLAST MCDB 187
2 BLAST Basic Local Alignment Sequence Tool Uses shortcut to compute alignments of a sequence against a database very quickly Typically takes about a minute to align a sequence against a database of one million sequences Accurately computes the statistical significance of the alignment
3 One of most highly cited papers
4 Global vs Local Alignments In 1981 Smith and Waterman introduced a modification to the Needleman-Wunsch algorithm to search for optimal alignment fragments Positions in the alignment matrix with negative scores are set to zero, and are considered new start positions
5 Definitions Maximum Segment Pair (MSP): highest scoring pair of identical length segments from 2 sequences Word pair: segment pair of fixed length w T: minimum word pair score
6 Algorithm Pre-compute all words that score above T against the query sequence Search for these words in a database For each match, extend the alignment to generate the maximum segment pair Compute statistics of the resulting score
7 Step 1 : break up sequence into words ATCGTCTATTCCCGG w=4 letter words ATCG TCGT CGTC etc.
8 Step 2: find high scoring matches Start with word 1: ATCG score identities as +1, and mismatches a 0 find all words that have a score of at least T=3 ATCA ATCT ATCC etc.
9 Search for high scoring words in database Find all sequences in the database that contain a high scoring word For example: CCGGCTATCATCTATTCCCGGTTCG
10 Extend ALignments Around matching word CCGGCTATCATCTATTCCCGGTTCG ATCGTCTATTCCCGG The Yellow seqeunce alignmnet corresponds to the MSP wth score S
11 Lecture
12 Different Flavours of BLAST BLASTP - protein query against protein DB BLASTN - DNA/RNA query against GenBank (DNA) BLASTX - 6 frame trans. DNA query against proteindb TBLASTN - protein query against 6 frame GB transl. TBLASTX - 6 frame DNA query to 6 frame GB transl. PSI-BLAST - protein profile query against protein DB PHI-BLAST - protein pattern against protein DB Lecture
13 Running NCBI BLAST Click BLAST! Lecture
14 MT0895 MMKIQIYGTGCANCQMLEKNAREAVKELGID AEFEKIKEMDQILEAGLTALPGLAVDGELKI MGRVASKEEIKKILS Lecture
15 Formatting Results Lecture
16 BLAST Format Options Lecture
17 BLAST Output Lecture
18 BLAST Output Lecture
19 BLAST Output Lecture
20 BLAST Output Lecture
21 BLAST Output Lecture
22 BLAST Output Lecture
23 BLAST Parameters Identities - No. & % exact residue matches Positives - No. and % similar & ID matches Gaps - No. & % gaps introduced Score - Summed HSP score (S) Bit Score - a normalized score (S ) Expect (E) - Expected # of chance HSP aligns P - Probability of getting a score > X T - Minimum word or k-tuple score (Threshold) Lecture
24 BLAST - Rules of Thumb Expect (E-value) is equal to the number of BLAST alignments with a given Score that are expected to be seen simply due to chance Don t trust a BLAST alignment with an Expect score > 0.01 (Grey zone is between ) Expect and Score are related, but Expect contains more information. Note that %Identies is more useful than the bit Score Recall Doolittle s Curve (%ID vs. Length, next slide) %ID > 30 - numres/50 If uncertain about a hit, perform a PSI-BLAST search Lecture
25 Doolittle s Curve 100 Evolutionary Distance VS Percent Sequence Identity Sequence Identity (%) Number of Residues Twilight Zone Lecture
26 Statistics Probability of observing a score greater than S Probability of observing multiple MSPs greater than S
27 Extreme Value Distribution
28 Scoring Matrices BLOSUM Matrices Developed by Henikoff & Henikoff (1992) BLOcks SUbstitution Matrix Derived from the BLOCKS database PAM Matrices Developed by Schwarz and Dayhoff (1978) Point Accepted Mutation Derived from manual alignments of closely related proteins Lecture
29 Conclusions BLAST is the most important program in bioinformatics (maybe all of biology) BLAST is based on sound statistical principles (key to its speed and sensitivity) A basic understanding of its principles is key for using/interpreting BLAST output Use NBLAST or MEGABLAST for DNA Use PSI-BLAST for protein searches Lecture
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