1. Introduction to Bioinformatics - Tutorial no. 2 BLAST

3. BLAST – Outline Sequence Alignment Complexity and indexing BLASTN and BLASTP  Basic parameters PAM and BLOSUM matrices Affine gap model E Values (once again)

4. Advanced BLAST Databases BLAST options BLAST output Taxonomic BLAST Pairwise BLAST

5. NameQuery typeDatabase blastnGenomic blastpProtein blastxTranslated genomicProtein tblastnProteinTranslated genomic tblastxTranslated genomic Genomic translations test all 6 possibilities: 3x for codon frames, 2x for reverse complement BLAST Variations

7. BLASTN Databases nr GenBank, EMBL, DDBJ, PDB and NCBI reference sequences (RefSeq) htgsHigh-throughput genomic sequences (draft) patPatented nucleotide sequences mitoMitochondrial sequences vectorVector subset of GenBank monthGenBank, EMBL, DDBJ, PDB from 30 days chromContigs and chromosomes from RefSeq

8. BLASTP Databases nr GenBank CDS translations, RefSeq, PDB, SWISS-PROT, PIR, PRF swissprotSWISS-PROT patPatented protein sequences pdbProtein Data Bank month GenBank CDS translations, PDB, SWISS-PROT, PIR, PRF from 30 days

9. BLASTN/P Options (1) Only search part of database using NCBI Entrez query format Search specific organism Remove low information content, e.g. short repeats or rich in only 2 nucleotides Remove known human repeats (LINEs, SINEs)

10. BLASTN/P Options (2) Threshold for results significance Use index based on words of 7, 11 or 15 nucleotides Costs to open and extend gap, score for nucleotide match or mismatch. Allowed gap scores: 10/1, 10/2, 11/1, 8/2, 9/2

11. BLASTP Options Scoring matrix: PAM, etc… Search for a motif (PSI-BLAST) Costs to open and extend gap

12. BLASTN/P Formatting (1) Show colored bar chart Number of sequences listed Number of alignments shown Other (less important) options on what to show

13. BLASTN/P Formatting (2) How to display alignments Only show results which match Entrez search or are from specific organism Only show results with E values in this range

14. BLASTN Results Query sequence representation Matched areas of database sequences

15. BLAST Output Header Request ID for later retrieval Query sequence details Database details Tax BLAST

16. BLAST Alignments (1) Sequence Identifier Sequence description Score and E value

17. BLAST Alignments (2) Normalized score of alignment Expected number of such hits (2e-11 = 2  10 -11 ) Number of exact matches Number of matches with positive score Number of insertion / deletions

18. BLAST Alignments (3) Query sequenceExact matchInsertion / deletion Matched sequence Mismatch with positive score Position within sequence Masked low complexity region

19. Expectation Values Increases linearly with length of query sequence Increases linearly with length of database Decreases exponentially with score of alignment

20. Tax BLAST Lineage of organism with strongest hit Score of organism’s strongest hit Number of organism hits Shared ancestry in taxonomic tree

21. BLAST2SEQ Scoring scheme Type of program Gap model, Expect Value, Advanced options Sequences Scoring matrix Sequences GO ! This tool produces the alignment of two given sequences using BLAST engine for local alignment.BLAST

22. Questions You have two query sequences: query1 and query2: >query1 CCGTCCGTCCGTCGTCCTCCTCGCTTGCGGGGCGCCGGGCCCGTCCTCGAGCCCCCNNNNNCCGTCCGGC CGCGTCGGGGCCTCGCCGCGCTCTACCTACCTACCTGGTTGATCCTGCCAGTAGCATATGCTTGTCTCAA AGATTAAGCCATGCATGTCTAAGTACGCACGGCCGGTACAGTGAAACTGCGAATGGCTCATTAAATCAGT TATGGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCC GACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCCC TCTCCGGCCCCGGCCGGGGGGCGGGCCGCGGCGGCTTTGGTGACTCTAGATAACCTCGGGCCGATCGCAC GCCCCCCGTGGCGGCGACGACCCATTCGAACGTCTGCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTA CCATGGTGACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCAC ATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCCCGACCCGGGGAGGTAGTGACGAAAAATAACAA TACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTCCACTTTAAATCCTTTAACGAGGATCCATTGGA GGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAA AAAGCTCGTAGTTGGATCTTGGGAGCGGGCGGGCGGTCCGCCGCGAGGCGAGCCACCGCCCGTCCCCGCC CCTTGCCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGTCCCGCGGGGCCCGAAGCGTTTACTTTGAA AAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATACCGCAGCTAGGAATAATGGAATAGGACCGC GGTTCTATTTTGTTGGTTTTCGGAACTGAGGCCATGATTAAGAGGGACGGCCGGGGGCATTCGTATTGCG CCGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGACCAGAGCGAAAGCATTTGCCAAGAATGTTTTC ATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATAAACGATG CCGACCGGCGATGCGGCGGCGTTATTCCCATGACCCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTGGG TTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAG CCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGACAGGATTGACAGATTGA TAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTA ATTCCGATAACGAACGAGACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCCAA CTTCTTAGAGGGACAAGTGGCGTTCAGCCACCCGAGATTGAGCAATAACAGGTCTGTGATGCCCTTAGAT GTCCGGGGCTGCACGCGCGCTACACTGACTGGCTCAGCGTGTGCCTACCCTACGCCGGCAGGCGCGGGTA ACCCGTTGAACCCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAATTCCCAGT AAGTGCGGGTCATAAGCTTGCGTTGATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGA TTGGATGGTTTAGTGAGGCCCTCGGATCGGCCCCGCCGGGGTCGGCCCACGGCCTGGCGGAGCGCTGAGA AGACGGTCGAA

23. Questions >query2 TACGAACGCTGGCGGCATGCTAATACATGCAAGTCGAACGAGACCTTCGGGTCTAGTGGCGCACGGGTGG CTAACGCGTGGGAATCTGCCCTTGGGTTCGGAATAACTTCGGGAAACTGAAGCTAATACCGGATGATGAC GAAAGTCCAAAGATTTATCGCCCAGGGATGAGCCCGCGTAGGATTAGCTAGTTGGTGGGGTAAAGGCTCA CCAAGGCAACGATCCTTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTAGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGTG ATGAAGGCCTTAGGGTTGTAAAGCTCTTTTACCCGAGATGATAATGACAGTATCGGGAGAATAAGCTCCG GCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGAGCTAGCGTTGTTCGGAATTACTGGGCGTAAAG CGCACGTAGGCGGCGATTTAAGTCAGAGGTGAAAGCCCGGGCTCAACCCCGAACTGCCTTTGAGACTGGA TTGCTAGAATCTTGGAGAGGCGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAAC ACCAGTGCGAAGGCGGCTCGCTGGACAAGTATTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGAT TAGATACCCTGGTAGTCCACGCCGTAAACGATGATAACTAGCTGCCGGGGCACATGGTGTTTCGGTGGCG CACGTAACGCATTAAGTTATCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGG GCCTGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCGTTTGACATC CTCATCGCGGATTTCAGAGATGATTTCCTTCAGTTCGGCTGGATGAGTGACAGGTGCTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTGCCAGCAT TTAGTTGGGTACTCTAAAGGAACCGCCGGTGATAAGCCGGAGAAGGTGGGGATGACGTCAAGTCCTCATG GCCCTTACGCGCTGGGCTACACACGTGCTACAATGGCGACTACAGTGGGCTGCAACCGTGCGAGCGGTAG CTAATCTCCAAAAGTCGTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGGCGGAATCGCTAG TAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCGCCCGTCACACCATGGG ATTTGGATTCACCCGAAGGCACTGCGTTAACCCGCAAGGGAGACAGGTGACCACGGTGGGTTTAGAGACT GGGGTGAA

24. Questions Using BLASTN Find what do each one of these sequences code for.

25. Questions

26. To which organism each sequence is related? Do these sequences code for proteins? Pretend the information for answering previous questions is not available to you could you suggest a way to answer these questions anyway? BLAST X

27. Questions Look carefully at the e-value column of the first 50 results of each query. What can you learn about these sequences? Are these sequences generally conserved between other organisms? 5 last answers

28. Questions Use bl2seq to align the two query sequences. What can you say about the relation between them? Based does this last result make sense?

29. Questions You have two query sequences. >query3 ATGTCTGCTCCACAAGCCAAGATTTTGTCTCAAGCTCCAACTGAATTGGAATTACAAGTT GCTCAAGCTTTCGTTGAATTGGAAAATTCTTCTCCAGAATTGAAAGCTGAGTTGAGACCT TTGCAATTCAAGTCCATCAGAGAAGT >query4 GTATGTTATTAATTTGAATCTAAACTTAAGAATAATGGAGAGTAACAAAGGAAAAAAGTG TGAACGGGACGATACCAGAATGTTTCAATCTAGAAAAGTATAAAAGATAAGGACTAGGAC TCAAATGTATTTGGCTGACTATCGCCTGAACCTTGATGCTAAGCAAATACCATATCTTCA AGAAAAAGCCTACTCCAGTGTTTAAGAAGAAGGGAACGATTTACTAGATCATGCTATACG CAGTAAGGTTCTGATAGTTAATTACAATCGGTCCAAGTTCTAAGCGGTGTCGTCCATGCA TATATCATTTACAAGTTACTGGCGTCAACTCTTCAAATATTCAAAATATCACCTAATCAA ACTTACTAACATTTTCCTTTTTTGTTTTCCTTCTTTTATAG Now use BlastX To what protein does these sequences code for? are these proteins conserved in other organisms?

30. Questions Now use BlastX To what protein does these sequences code for? are these proteins conserved in other organisms? A conserved protein component of the small (40S) subunit of S. cerevisiae. Query 3Query 4 No protein – e-value 3.2

31. Questions You are told that the sequences were extracted from the same gene. How could you explain the above results? Answer: query4 is extracted from a non-coding region (intron) and thus doesn’t code for any protein.