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CAP5510 – BioinformaticsDatabase Searches for Biological Sequences or Imperfect Alignments

Tamer KahveciCISE Department

University of Florida

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Goals

• Understand how major heuristic methods for sequence comparison work– FASTA– BLAST

• Understand how search results are evaluated

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What is Database Search ?• Find a particular (usually) short sequence in a

database of sequences (or one huge sequence).• Problem is identical to local sequence alignment,

but on a much larger scale.• We must also have some idea of the significance

of a database hit.– Databases always return some kind of hit, how much

attention should be paid to the result?• A similar problem is the global alignment of two

large sequences• General idea: good alignments contain high

scoring regions.

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Imperfect Alignment

• What is an imperfect alignment?• Why imperfect alignment?

• The result may not be optimal.• Finding optimal alignment is usually

to costly in terms of time and memory.

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Database Search Methods

• Hash table based methods– FASTA family

• FASTP, FASTA, TFASTA, FASTAX, FASTAY

– BLAST family• BLASTP, BLASTN, TBLAST, BLASTX, BLAT, BLASTZ,

MegaBLAST, PsiBLAST, PhiBLAST

– Others• FLASH, PatternHunter, SSAHA, SENSEI, WABA, GLASS

• Suffix tree based methods– Mummer, AVID, Reputer, MGA, QUASAR

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History of sequence searching

• 1970: NW• 1980: SW• 1985: FASTA• 1990: BLAST

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Hash Table

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Hash Table

• K-gram = subsequence of length K

• Ak entries– A is alphabet

size

• Linear time construction

• Constant lookup time

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FASTP

Lipman & Pearson, 1985

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FASTP

• Three phase algorithm1. Find short good matches using k-

grams1. K = 1 or 2

2. Find start and end positions for good matches

3. Use DP to align good matches

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position 1 2 3 4 5 6 7 8 9 10 11protein 1 n c s p t a . . . . . protein 2 . . . . . a c s p r k position in offsetamino acid protein A protein B pos A - posB-----------------------------------------------------a 6 6 0c 2 7 -5k - 11n 1 -p 4 9 -5r - 10s 3 8 -5t 5 ------------------------------------------------------Note the common offset for the 3 amino acids c,s and pA possible alignment can be quickly found :protein 1 n c s p t a | | | protein 2 a c s p r k

FASTP: Phase 1 (1)

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FASTP: Phase 1 (2)• Similar to dot plot• Offsets range from 1-

m to n-1• Each offset is scored

as – # matches - #

mismatches• Diagonals (offsets)

with large score show local similarities

• How does it depend on k?

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FASTP: Phase 2

• 5 best diagonal runs are found

• Rescore these 5 regions using PAM250.– Initial score

• Indels are not considered yet

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FASTP: Phase 3

• Sort the aligned regions in descending score

• Optimize these alignments using Needleman-Wunsch

• Report the results

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FASTP - Discussion

• Results are not optimal. Why ?

• How does performance compare to Smith-Waterman?

• What is the impact of k?

• How does this idea work for DNAs ?– K = 4 or 6 for DNA

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FASTA – Improvement Over FASTP

Pearson 1995

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FASTA (1)

• Phase 2: Choose 10 best diagonal runs instead of 5

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FASTA (2)• Phase 2.5

– Eliminate diagonals that score less than some given threshold.

– Combine matches to find longer matches. It incurs join penalty similar to gap penalty

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FASTA Variations

• TFASTAX and TFASTAY: query protein against a DNA library in all reading frames

• FASTAX, FASTAY: DNA query in all reading frames against protein database

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BLAST

Altschul, Gish, Miller, Myers, Lipman, 1990

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BLAST (or BLASTP)

• BLAST – Basic Local Alignment Search Tool

• An approximation of Smith-Waterman

• Designed for database searches– Short query sequence against long

database sequence or a database of many sequences

• Sacrifices search sensitivity for speed

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BLAST Algorithm (1)

• Eliminate low complexity regions from the query sequence.– Replace them with X (protein) or N

(DNA)• Hash table on query sequence.

– K = 3 for proteins

MCG

CGP

MCGPFILGTYC

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BLAST Algorithm (2)

• For each k-gram find all k-grams that align with score at least cutoff T using BLOSUM62– 20k candidates– ~50 on the average per

k-gram– ~50n for the entire

query

• Build hash table

PQG

QGM

PQGMCGPFILGTYC

PQGPQG 18PEG 15PRG 14PSG 13PQA 12

T = 13

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BLAST Algorithm (3)

• Sequentially scan the database and locate each k-gram in the hash table

• Each match is a seed for an ungapped alignment.

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BLAST Algorithm (4)

• HSP (High Scoring Pair) = A match between a query word and the database

• Find a “hit”: Two non-overlapping HSP’s on a diagonal within distance A

• Extend the hit until the score falls below a threshold value, X

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BLAST Algorithm (5)

• Keep only the extended matches that have a score at least S.

• Determine the statistical significance of the result

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What is Statistical Significance?

13 : 15

13 : 15

•Two one-on-one games, two scores.

•Which result is more significant?

•Expected: maybe a random result.•Unexpected: significant, may have significant meanings.

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Statistical Significance

• E-value: The expected number of matches with score at least S

• E = Kmne-lambda.S

• m, n : sequence lengths• S : alignment score• K, lambda: normalization parameters

• P-value: The probability of having at least one match with score at least S

• 1 – e-E

• The smaller these values are, the more significant the result

• http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/glossary2.html

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BLAST - Analysis

• K (k-gram)– Lower: more sensitive.

Slower.

• T (neighbor cutoff)– Lower: Find distant

neighbors. Introduces noise

• X (extension cutoff)– Higher: lower chances

of getting into a local minima. Slower.

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Sample Query

• http://www.ncbi.nlm.nih.gov/BLAST/

I D R A M S A A R G V F E R G D W S L S S P A K R K A V L N K L A D L M E A H A E E L A L L E T L D T G K P I R H S L R D D I P G A A R A I R W Y A E A I D K V Y G E V A T T S S H E L A M I V R E P V G V I A A I V P W N F P L L L T C W K L G P A L A A G N S V I L K P S E K S P L S A I R L A G L A K E A G L P D G V L N V V T G F G H E A G Q A L S R H N D I D A I A F T G S T R T G K Q L L K D A G D S N M K R V W L E A G G K S A N I V F A D C P D L Q Q A A S A T A A G I F Y N Q G Q V C I A G T R L L L E E S I A D E F L A L L K Q Q A Q N W Q P G H P L D P A T T M G T L I D C A H A D S V H S F I R E G E S K G Q L L L D G R N A G L A A A I G P T I F V D V D P N A S L S R E E I F G P V L V V T R F T S E E Q A L Q L A N D S Q Y G L G A A V W T R D L S R A H R M S R R L K A G S V F V N N Y N D G D M T V P F G G Y K Q S G N G R D K S L H A L E K F T E L K T I W I

Dhal_ecoli

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BLASTN

• BLAST for nucleic acids• K = 11• Exact match instead of neighborhood

search.

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BLAST Variations

Program Query Target Type

BLASTP Protein Protein Gapped

BLASTN Nucleic acid Nucleic acid Gapped

BLASTX Nucleic acid Protein Gapped

TBLASTN Protein Nucleic acid Gapped

TBLASTX Protein Nucleic acid Gapped

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Even More Variations

– PsiBLAST (iterative)– BLAT, BLASTZ, MegaBLAST– FLASH, PatternHunter, SSAHA, SENSEI,

WABA, GLASS

– Main differences are• Seed choice (k, gapped seeds)• Additional data structures

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Suffix Trees

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Suffix Tree• Tree structure that contains all suffixes of the input

sequence

• TGAGTGCGA• GAGTGCGA• AGTGCGA• GTGCGA• TGCGA• GCGA• CGA• GA• A

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Suffix Tree Example

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• O(n) space and construction time– 10n to 70n space usage reported

• O(m) search time for m-letter sequence

• Good for – Small data– Exact matches

Suffix Tree Analysis

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Suffix Array

• 5 bytes per letter• O(m log n) search

time

• Better space usage• Slower search

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Mummer

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Other Sequence Comparison Tools

• Reputer, MGA, AVID• QUASAR (suffix array)