Blast fasta 4
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BLAST and FASTA are algorithms for searching sequence databases to find local alignments between a query sequence and database sequences, with BLAST providing faster searches and improved statistical analysis compared to FASTA. Both algorithms work by first identifying short exact matches between sequences and then extending these matches to identify longer regions of similarity. The algorithms model DNA and protein sequence alignments as coin tosses to determine the expected length of the longest matching region between random sequences.
![FASTA Format
• simple format used by almost all programs
• >header line with a [return] at end
• Sequence (no specific requirements for line
length, characters, etc)
>URO1 uro1.seq Length: 2018 November 9, 2000 11:50 Type: N Check: 3854 ..
CGCAGAAAGAGGAGGCGCTTGCCTTCAGCTTGTGGGAAATCCCGAAGATGGCCAAAGACA
ACTCAACTGTTCGTTGCTTCCAGGGCCTGCTGATTTTTGGAAATGTGATTATTGGTTGTT
GCGGCATTGCCCTGACTGCGGAGTGCATCTTCTTTGTATCTGACCAACACAGCCTCTACC
CACTGCTTGAAGCCACCGACAACGATGACATCTATGGGGCTGCCTGGATCGGCATATTTG
TGGGCATCTGCCTCTTCTGCCTGTCTGTTCTAGGCATTGTAGGCATCATGAAGTCCAGCA
GGAAAATTCTTCTGGCGTATTTCATTCTGATGTTTATAGTATATGCCTTTGAAGTGGCAT
CTTGTATCACAGCAGCAACACAACAAGACTTTTTCACACCCAACCTCTTCCTGAAGCAGA
TGCTAGAGAGGTACCAAAACAACAGCCCTCCAAACAATGATGACCAGTGGAAAAACAATG
GAGTCACCAAAACCTGGGACAGGCTCATGCTCCAGGACAATTGCTGTGGCGTAAATGGTC
CATCAGACTGGCAAAAATACACATCTGCCTTCCGGACTGAGAATAATGATGCTGACTATC
CCTGGCCTCGTCAATGCTGTGTTATGAACAATCTTAAAGAACCTCTCAACCTGGAGGCTT 22](https://image.slidesharecdn.com/blastfasta-4-121128073311-phpapp02/85/Blast-fasta-4-22-320.jpg)
![BLAST Searches GenBank
[BLAST= Basic Local Alignment Search Tool]
The NCBI BLAST web server lets you compare your
query sequence to various sections of GenBank:
– nr = non-redundant (main sections)
– month = new sequences from the past few weeks
– refseq_rna
– RNA entries from NCBI's Reference Sequence project
– refseq_genomic
– Genomic entries from NCBI's Reference Sequence project
– ESTs
– Taxon = e.g., human, Drososphila, yeast, E. coli
– proteins (by automatic translation)
– pdb = Sequences derived from the 3-dimensional structure
from Brookhaven Protein Data Bank
29](https://image.slidesharecdn.com/blastfasta-4-121128073311-phpapp02/85/Blast-fasta-4-29-320.jpg)
Blast fasta 4
- 1. BLAST and FASTA 1
- 2. Pairwise Alignment Global Local • Best score from among • Best score from among alignments of full-length alignments of partial sequences sequences • Needelman-Wunch • Smith-Waterman algorithm algorithm 2
- 3. Why do we need local alignments? • To compare a short sequence to a large one. • To compare a single sequence to an entire database • To compare a partial sequence to the whole. 3
- 4. Why do we need local alignments? • Identify newly determined sequences • Compare new genes to known ones • Guess functions for entire genomes full of ORFs of unknown function 4
- 5. Mathematical Basis for Local Alignment • Model matches as a sequence of coin tosses • Let p be the probability of “head” – For a “fair” coin, p = 0.5 • According to Paul Erdös-Alfréd Rényi law: If there are n throws, then the expected length, R, of the longest run of “heads” is R = log1/p (n). Paul Erdös 5
- 6. Mathematical Basis for Local Alignment • Example: Suppose n = 20 for a “fair” coin R=log2(20)=4.32 • Problem: How does one model DNA (or amino acid) alignments as coin tosses. 6
- 7. Modeling Sequence Alignments • To model random sequence alignments, replace a match by “head” (H) and mismatch by “tail” (T). AATCAT HTHHHT ATTCAG • For ungapped DNA alignments, the probability of a “head” is 1/4. • For ungapped amino acid alignments, the probability of a “head” is 1/20. 7
- 8. Modeling Sequence Alignments • Thus, for any one particular alignment, the Erdös- Rényi law can be applied • What about for all possible alignments? – Consider that sequences can being shifted back and forth in the dot matrix plot • The expected length of the longest match is R = log1/p(mn) where m and n are the lengths of the two sequences. 8
- 9. Modeling Sequence Alignments • Suppose m = n = 10, and we deal with DNA sequences R = log4(100) = 3.32 • This analysis assumes that the base composition is uniform and the alignment is ungapped. The result is approximate, but not bad. 9
- 10. 10
- 11. Heuristic Methods: FASTA and BLAST FASTA • First fast sequence searching algorithm for comparing a query sequence against a database. BLAST • Basic Local Alignment Search Technique improvement of FASTA: Search speed, ease of use, statistical rigor. 11
- 12. FASTA and BLAST • Basic idea: a good alignment contains subsequences of absolute identity (short lengths of exact matches): – First, identify very short exact matches. – Next, the best short hits from the first step are extended to longer regions of similarity. – Finally, the best hits are optimized. 12
- 13. FASTA Derived from logic of the dot plot – compute best diagonals from all frames of alignment The method looks for exact matches between words in query and test sequence – DNA words are usually 6 nucleotides long – protein words are 2 amino acids long 13
- 14. FASTA Algorithm 14
- 15. Makes Longest Diagonal After all diagonals are found, tries to join diagonals by adding gaps Computes alignments in regions of best diagonals 15
- 16. FASTA Alignments 16
- 17. FASTA Results - Histogram !!SEQUENCE_LIST 1.0 (Nucleotide) FASTA of: b2.seq from: 1 to: 693 December 9, 2002 14:02 TO: /u/browns02/Victor/Search-set/*.seq Sequences: 2,050 Symbols: 913,285 Word Size: 6 Searching with both strands of the query. Scoring matrix: GenRunData:fastadna.cmp Constant pamfactor used Gap creation penalty: 16 Gap extension penalty: 4 Histogram Key: Each histogram symbol represents 4 search set sequences Each inset symbol represents 1 search set sequences z-scores computed from opt scores z-score obs exp (=) (*) < 20 0 0: 22 0 0: 24 3 0:= 26 2 0:= 28 5 0:== 30 11 3:*== 32 19 11:==*== 34 38 30:=======*== 36 58 61:===============* 38 79 100:==================== * 40 134 140:==================================* 42 167 171:==========================================* 44 205 189:===============================================*==== 46 209 192:===============================================*===== 17 48 177 184:=============================================*
- 18. FASTA Results - List The best scores are: init1 initn opt z-sc E(1018780).. SW:PPI1_HUMAN Begin: 1 End: 269 ! Q00169 homo sapiens (human). phosph... 1854 1854 1854 2249.3 1.8e-117 SW:PPI1_RABIT Begin: 1 End: 269 ! P48738 oryctolagus cuniculus (rabbi... 1840 1840 1840 2232.4 1.6e-116 SW:PPI1_RAT Begin: 1 End: 270 ! P16446 rattus norvegicus (rat). pho... 1543 1543 1837 2228.7 2.5e-116 SW:PPI1_MOUSE Begin: 1 End: 270 ! P53810 mus musculus (mouse). phosph... 1542 1542 1836 2227.5 2.9e-116 SW:PPI2_HUMAN Begin: 1 End: 270 ! P48739 homo sapiens (human). phosph... 1533 1533 1533 1861.0 7.7e-96 SPTREMBL_NEW:BAC25830 Begin: 1 End: 270 ! Bac25830 mus musculus (mouse). 10, ... 1488 1488 1522 1847.6 4.2e-95 SP_TREMBL:Q8N5W1 Begin: 1 End: 268 ! Q8n5w1 homo sapiens (human). simila... 1477 1477 1522 1847.6 4.3e-95 SW:PPI2_RAT Begin: 1 End: 269 ! P53812 rattus norvegicus (rat). pho... 1482 1482 1516 1840.4 1.1e-94 18
- 19. FASTA Results - Alignment SCORES Init1: 1515 Initn: 1565 Opt: 1687 z-score: 1158.1 E(): 2.3e-58 >>GB_IN3:DMU09374 (2038 nt) initn: 1565 init1: 1515 opt: 1687 Z-score: 1158.1 expect(): 2.3e-58 66.2% identity in 875 nt overlap (83-957:151-1022) 60 70 80 90 100 110 u39412.gb_pr CCCTTTGTGGCCGCCATGGACAATTCCGGGAAGGAAGCGGAGGCGATGGCGCTGTTGGCC || ||| | ||||| | ||| ||||| DMU09374 AGGCGGACATAAATCCTCGACATGGGTGACAACGAACAGAAGGCGCTCCAACTGATGGCC 130 140 150 160 170 180 120 130 140 150 160 170 u39412.gb_pr GAGGCGGAGCGCAAAGTGAAGAACTCGCAGTCCTTCTTCTCTGGCCTCTTTGGAGGCTCA ||||||||| || ||| | | || ||| | || || ||||| || DMU09374 GAGGCGGAGAAGAAGTTGACCCAGCAGAAGGGCTTTCTGGGATCGCTGTTCGGAGGGTCC 190 200 210 220 230 240 180 190 200 210 220 230 u39412.gb_pr TCCAAAATAGAGGAAGCATGCGAAATCTACGCCAGAGCAGCAAACATGTTCAAAATGGCC ||| | ||||| || ||| |||| | || | |||||||| || ||| || DMU09374 AACAAGGTGGAGGACGCCATCGAGTGCTACCAGCGGGCGGGCAACATGTTTAAGATGTCC 250 260 270 280 290 300 240 250 260 270 280 290 u39412.gb_pr AAAAACTGGAGTGCTGCTGGAAACGCGTTCTGCCAGGCTGCACAGCTGCACCTGCAGCTC |||||||||| ||||| | |||||| |||| ||| || ||| || | DMU09374 AAAAACTGGACAAAGGCTGGGGAGTGCTTCTGCGAGGCGGCAACTCTACACGCGCGGGCT 19 310 320 330 340 350 360
- 20. FASTA on the Web • Many websites offer FASTA searches • Each server has its limits • Be aware that you depend “on the kindness of strangers.” 20
- 21. Institut de Génétique Humaine, Montpellier France, GeneStream server http://www2.igh.cnrs.fr/bin/fasta-guess.cgi Oak Ridge National Laboratory GenQuest server http://avalon.epm.ornl.gov/ European Bioinformatics Institute, Cambridge, UK http://www.ebi.ac.uk/htbin/fasta.py?request EMBL, Heidelberg, Germany http://www.embl-heidelberg.de/cgi/fasta-wrapper-free Munich Information Center for Protein Sequences (MIPS) at Max-Planck-Institut, Germany http://speedy.mips.biochem.mpg.de/mips/programs/fasta.html Institute of Biology and Chemistry of Proteins Lyon, France http://www.ibcp.fr/serv_main.html Institute Pasteur, France http://central.pasteur.fr/seqanal/interfaces/fasta.html GenQuest at The Johns Hopkins University http://www.bis.med.jhmi.edu/Dan/gq/gq.form.html National Cancer Center of Japan http://bioinfo.ncc.go.jp 21
- 22. FASTA Format • simple format used by almost all programs • >header line with a [return] at end • Sequence (no specific requirements for line length, characters, etc) >URO1 uro1.seq Length: 2018 November 9, 2000 11:50 Type: N Check: 3854 .. CGCAGAAAGAGGAGGCGCTTGCCTTCAGCTTGTGGGAAATCCCGAAGATGGCCAAAGACA ACTCAACTGTTCGTTGCTTCCAGGGCCTGCTGATTTTTGGAAATGTGATTATTGGTTGTT GCGGCATTGCCCTGACTGCGGAGTGCATCTTCTTTGTATCTGACCAACACAGCCTCTACC CACTGCTTGAAGCCACCGACAACGATGACATCTATGGGGCTGCCTGGATCGGCATATTTG TGGGCATCTGCCTCTTCTGCCTGTCTGTTCTAGGCATTGTAGGCATCATGAAGTCCAGCA GGAAAATTCTTCTGGCGTATTTCATTCTGATGTTTATAGTATATGCCTTTGAAGTGGCAT CTTGTATCACAGCAGCAACACAACAAGACTTTTTCACACCCAACCTCTTCCTGAAGCAGA TGCTAGAGAGGTACCAAAACAACAGCCCTCCAAACAATGATGACCAGTGGAAAAACAATG GAGTCACCAAAACCTGGGACAGGCTCATGCTCCAGGACAATTGCTGTGGCGTAAATGGTC CATCAGACTGGCAAAAATACACATCTGCCTTCCGGACTGAGAATAATGATGCTGACTATC CCTGGCCTCGTCAATGCTGTGTTATGAACAATCTTAAAGAACCTCTCAACCTGGAGGCTT 22
- 23. Assessing Alignment Significance • Generate random alignments and calculate their scores • Compute the mean and the standard deviation (SD) for random scores • Compute the deviation of the actual score from the mean of random scores Z = (meanX)/SD • Evaluate the significance of the alignment • The probability of a Z value is called the E score 23
- 24. E scores or E values E scores are not equivalent to p values where p < 0.05 are generally considered statistically significant. 24
- 25. E values (rules of thumb) E values below 10-6 are most probably statistically significant. E values above 10-6 but below 10-3 deserve a second look. E values above 10-3 should not be tossed aside lightly; they should be thrown out with great force. 25
- 26. BLAST • Basic Local Alignment Search Tool – Altschul et al. 1990,1994,1997 • Heuristic method for local alignment • Designed specifically for database searches • Based on the same assumption as FASTA that good alignments contain short lengths of exact matches 26
- 27. BLAST • Both BLAST and FASTA search for local sequence similarity - indeed they have exactly the same goals, though they use somewhat different algorithms and statistical approaches. • BLAST benefits – Speed – User friendly – Statistical rigor – More sensitive 27
- 28. Input/Output • Input: – Query sequence Q – Database of sequences DB – Minimal score S • Output: – Sequences from DB (Seq), such that Q and Seq have scores > S 28
- 29. BLAST Searches GenBank [BLAST= Basic Local Alignment Search Tool] The NCBI BLAST web server lets you compare your query sequence to various sections of GenBank: – nr = non-redundant (main sections) – month = new sequences from the past few weeks – refseq_rna – RNA entries from NCBI's Reference Sequence project – refseq_genomic – Genomic entries from NCBI's Reference Sequence project – ESTs – Taxon = e.g., human, Drososphila, yeast, E. coli – proteins (by automatic translation) – pdb = Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank 29
- 30. BLAST • Uses word matching like FASTA • Similarity matching of words (3 amino acids, 11 bases) – does not require identical words. • If no words are similar, then no alignment – Will not find matches for very short sequences • Does not handle gaps well • “gapped BLAST” is somewhat better 30
- 31. BLAST Algorithm 31
- 32. BLAST Word Matching MEAAVKEEISVEDEAVDKNI MEA EAA AAV Break query AVK VKE into words: KEE EEI EIS ISV ... Break database sequences into words: 32
- 33. Find locations of matching words in database sequences ELEPRRPRYRVPDVLVADPPIARLSVSGRDENSVELT MEAT MEA EAA TDVRWMSETGIIDVFLLLGPSISDVFRQYASLTGTQALPPLFSLGYHQSRWNY AAV IWLDIEEIHADGKRYFTWDPSRFPQPRTMLERLASKRRV KLVAIVDPH AVK KLV KEE EEI EIS ISV 33
- 34. Extend hits one base at a time 34
- 35. Seq_XYZ: HVTGRSAF_FSYYGYGCYCGLGTGKGLPVDATDRCCWA Query: QSVFDYIYYGCYCGWGLG_GK__PRDA E-val=10-13 •Use two word matches as anchors to build an alignment between the query and a database sequence. •Then score the alignment. 35
- 36. HSPs are Aligned Regions • The results of the word matching and attempts to extend the alignment are segments - called HSPs (High-Scoring Segment Pairs) • BLAST often produces several short HSPs rather than a single aligned region 36
- 37. • >gb|BE588357.1|BE588357 194087 BARC 5BOV Bos taurus cDNA 5'. • Length = 369 • Score = 272 bits (137), Expect = 4e-71 • Identities = 258/297 (86%), Gaps = 1/297 (0%) • Strand = Plus / Plus • • Query: 17 aggatccaacgtcgctccagctgctcttgacgactccacagataccccgaagccatggca 76 • |||||||||||||||| | ||| | ||| || ||| | |||| ||||| ||||||||| • Sbjct: 1 aggatccaacgtcgctgcggctacccttaaccact-cgcagaccccccgcagccatggcc 59 • • Query: 77 agcaagggcttgcaggacctgaagcaacaggtggaggggaccgcccaggaagccgtgtca 136 • |||||||||||||||||||||||| | || ||||||||| | ||||||||||| ||| || • Sbjct: 60 agcaagggcttgcaggacctgaagaagcaagtggagggggcggcccaggaagcggtgaca 119 • • Query: 137 gcggccggagcggcagctcagcaagtggtggaccaggccacagaggcggggcagaaagcc 196 • |||||||| | || | ||||||||||||||| ||||||||||| || |||||||||||| • Sbjct: 120 tcggccggaacagcggttcagcaagtggtggatcaggccacagaagcagggcagaaagcc 179 • • Query: 197 atggaccagctggccaagaccacccaggaaaccatcgacaagactgctaaccaggcctct 256 • ||||||||| | |||||||| |||||||||||||||||| |||||||||||||||||||| • Sbjct: 180 atggaccaggttgccaagactacccaggaaaccatcgaccagactgctaaccaggcctct 239 • • Query: 257 gacaccttctctgggattgggaaaaaattcggcctcctgaaatgacagcagggagac 313 • || || ||||| || ||||||||||| | |||||||||||||||||| |||||||| • Sbjct: 240 gagactttctcgggttttgggaaaaaacttggcctcctgaaatgacagaagggagac 296 37
- 38. BLAST variants 38
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- 43. 43
- 44. Understanding BLAST output 44
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- 54. Choosing the right parameters 54
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- 57. 57
- 58. Controlling the output 58
- 59. 59
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- 63. More on BLAST NCBI Blast Glossary http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/glossary2.html Education: Blast Information http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html Steve Altschul's Blast Course http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html 63