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BINF350, Tutorial 4 Karen Marshall

Blast in practice. BINF350, Tutorial 4 Karen Marshall. Aim. Examine how blast parameters (e.g. scoring scheme, word length) affect the alignment outcome To optimise blast parameters for alignments with different levels of sequence homology. v. Practical: Part 1.

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BINF350, Tutorial 4 Karen Marshall

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  1. Blast in practice BINF350, Tutorial 4 Karen Marshall

  2. Aim • Examine how blast parameters (e.g. scoring scheme, word length) affect the alignment outcome • To optimise blast parameters for alignments with different levels of sequence homology

  3. v Practical: Part 1 • Start with an ~200 bp original DNA sequence • Simulation mutation events over time and collect sequences • Blast original sequence against mutated sequences • Repeat blasts using different parameters Mutated sequences Blast Original sequence

  4. Simulation of mutated sequences • Point accepted mutation (PAM) model of molecular evolution • 1 PAM = 1 mutation per 100 bases on average • 1 PAM  99.0% sequence homology • 10 PAM  90.6% sequence homology • 50 PAM  63.5% sequence homology • Concept of forward and backwards mutation

  5. for each ‘successive PAM’ for each ‘nucleotide’ if (rand > 0.01) do not mutate else if (rand <=0.01) mutate by random selection from the non-identical bases

  6. Step 1 2 3 BLAST - Heuristic • Suffix Tree • Lookup table • Words/seeds • Location • Threshold T • Larger seq file

  7. BLAST http://www.ncbi.nih.gov/BLAST/blast_whatsnew.shtml • BLAST 2.2.8 release notes • Correction to tblastx alignment computation • ia32-linux now requires glibc 2.2.5 • Source code can be obtained from: ftp://ftp.ncbi.nlm.nih.gov/toolbox/ncbi_tools/old/20040204/ncbi.tar.gz . • Binaries can be obtained from: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/2.2.8/ . • BLAST 2.2.7 release notes • Standalone BLAST is now available for amd64-linux. • formatdb now restricts volume sizes to 1G on 32-bit platforms for performance reasons. • The -A option has been removed from formatdb, that is, all databases will be created with ASN.1 deflines. • tblastn query concatenation now works correctly on 64-bit platforms. • The wwwblast source code has been merged into the C toolkit tree and is no longer distributed with the binaries. • Source code can be obtained from: ftp://ftp.ncbi.nlm.nih.gov/toolbox/ncbi_tools/old/20040202/ncbi.tar.gz . • Binaries can be obtained from: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/2.2.7/ .

  8. BLAST on your own machine • Allows you to BLAST multiple sequences • most web versions are single sequence only • Steps • Sequence files in FASTA format Can have multiple sequences in each file but no duplicates • Format larger sequence file into a database Formatdb –i dbfile.txt –p F –o T • Perform BLAST using appropriate switches BLASTALL –p BLASTN –d dbfile.txt –i comp.txt –o out.txt

  9. BLAST 2.2.8 • Arguments see appendix of handout • –W for seed word length (default = 11) • -r reward for a match (default = 1) • -q penalty for a mismatch (default = 3) • -G cost to open a gap • -E cost to extend a gap • -F filter query sequence • -e to set threshold expectation (threshold for HSP before gaps are included) • -m to specify different output options

  10. Example of BLAST output: -m3 Score E Sequences producing significant alignments: (bits) Value 1_10 170 3e-046 0_0 170 3e-046 4_10 115 2e-029 2_10 107 4e-027 5_10 96 2e-023 3_10 96 2e-023 4_20 68 3e-015 2_20 68 3e-015 5_20 56 1e-011 QUERY 1 agattcactggtgtggcaagttgtctctcagactgtacatgcattaaaattttgcttggc 60 1_10 1 ............................................................ 60 0_0 1 ............................................................ 60 4_10 3 ....t.....c......ag..................a.................... 60 2_10 1 ............a..c....a...........a................g.......... 60 5_10 2 ........c......a.........g............................c.... 60 3_10 1 .................g........t.....................c.....a..... 60 4_20 3 ....t.....c......ag....a.....g.......a.................... 60 2_20 1 ............a..c...ta...........aa......c..a.....g..... 55 5_20 4 ......c..c...a....g....g..............a......c......c.... 60

  11. Substitution scores • Optimal substitution scores were derived for different PAM distances / sequence homologies (States et al., 1991) • Of importance is the match to mismatch score ratio

  12. Substitution scores • ‘Better’ substitution matrices exist, but not yet implemented in most BLAST software

  13. Practical: Part 2 • Apply concepts from Part 1 to ‘real sequences’ • BLAST mRNA sequence for human and cattle INFG to an ~1/2 Mb sequence of human DNA • Use optimal blast parameters for expected homology Human DNA Blast Human INFG mRNA Cattle INFG mRNA

  14. Expected levels of sequence homology • Varies for sequences being considered and genomic region Human to mouse comparison, from …

  15. Efficiency of BLAST • Human to cattle coding sequence ~85% homology (~PAM 15)

  16. INFG mRNA sequences • Extracted from NCBI website using batch entrez INFG_refseq.txt >gi|10835170|ref|NM_000619.1| Homo sapiens interferon, gamma (IFNG), mRNA TGAAGATCAGCTATTAGAAGAGAAAGATCAGTTAAGTCCTTTGGACCTGATCAGCTTGATACAAGAACTA CTGATTTCAACTTCTTTGGCTTAATTCTCTCGGAAACGATGAAATATACAAGTTATATCTTGGCTTTTCA GCTCTGCATCGTTTTGGGTTCTCTTGGCTGTTACTGCCAGGACCCATATGTAAAAGAAGCAGAAAACCTT AAGAAATATTTTAATGCAGGTCATTCAGATGTAGCGGATAATGGAACTCTTTTCTTAGGCATTTTGAAGA ATTGGAAAGAGGAGAGTGACAGAAAAATAATGCAGAGCCAAATTGTCTCCTTTTACTTCAAACTTTTTAA AAACTTTAAAGATGACCAGAGCATCCAAAAGAGTGTGGAGACCATCAAGGAAGACATGAATGTCAAGTTT TTCAATAGCAACAAAAAGAAACGAGATGACTTCGAAAAGCTGACTAATTATTCGGTAACTGACTTGAATG TCCAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTGAACTGTCGCCAGCAGCTAAAACAGGGAA GCGAAAAAGGAGTCAGATGCTGTTTCAAGGTCGAAGAGCATCCCAGTAATGGTTGTCCTGCCTGCAATAT TTGAATTTTAAATCTAAATCTATTTATTAATATTTAACATTATTTATATGGGGAATATATTTTTAGACTC ATCAATCAAATAAGTATTTATAATAGCAACTTTTGTGTAATGAAAATGAATATCTATTAATATATGTATT ATTTATAATTCCTATATCCTGTGACTGTCTCACTTAATCCTTTGTTTTCTGACTAATTAGGCAAGGCTAT GTGATTACAAGGCTTTATCTCAGGGGCCAACTAGGCAGCCAACCTAAGCAAGATCCCATGGGTTGTGTGT TTATTTCACTTGATGATACAATGAACACTTATAAGTGAAGTGATACTATCCAGTTACTGCCGGTTTGAAA ATATGCCTGCAATCTGAGCCAGTGCTTTAATGGCATGTCAGACAGAACTTGAATGTGTCAGGTGACCCTG ATGAAAACATAGCATCTCAGGAGATTTCATGCCTGGTGCTTCCAAATATTGTTGACAACTGTGACTGTAC CCAAATGGAAAGTAACTCATTTGTTAAAATTATCAATATCTAATATATATGAATAAAGTGTAAGTTCACA ACT >gi|31982948|ref|NM_174086.1| Bos taurus interferon, gamma or immune type [interferon gamma type 2] (IFNG), mRNA ATTAGAAAAGAAAGATCAGCTACCTCCTTGGGACCTGATCATAACACAGGAGCTACCGATTTCAACTACT CCGGCCTAACTCTCTCCTAAACAATGAAATATACAAGCTATTTCTTAGCTTTACTGCTCTGTGGGCTTTT GGGTTTTTCTGGTTCTTATGGCCAGGGCCAATTTTTTAGAGAAATAGAAAACTTAAAGGAGTATTTTAAT GCAAGTAGCCCAGATGTAGCTAAGGGTGGGCCTCTCTTCTCAGAAATTTTGAAGAATTGGAAAGATGAAA

  17. Human Chr12 sub-sequence • Extracted from USCS ‘Golden Path’ website • chr12:66,589,493-67,085,092 ~ ½ Mb • does contain INFG gene • Repeats masked to lower case >hg16_dna range=chr12:66589493-67085092 5'pad=0 3'pad=0 revComp=FALSE strand=? repeatMasking=lower CATTCATTACTTTTATAAGGTTTCTCTCTGGTATGCATCTGACTTACATC ATGGGAAAGCTAGTTTCATGACTCCTTTGGAATAGTTGTGGTCCTGAATA TGGAAAATCAATTAATGAATAGCTTAAAGCACAATAGTCAACAAATAGAT GTGAAAATTCTTTGTGAACTTTAAAGTCTTACTTAAACGTGAGATATTAT ATACAGTGTTTTATGTtagactgtgagcttgttaaagaaagaactatgcc ttctttttctttctaccagttccagtgcctcgtacaacatagaaaccata agtgtttttgaaagagcaaatGAATATTGGAAGGAGTAAGGTGATAGCTA AAGCTAAAACAATGTTTAGGGAGAACAACTGAAACAAAAGCAGCATTTGT GTCTTAAACTCATGGCCTCTGAAACAGCCTTGATAGATAGTAGAGAGGGT CAGATAGAGAGAGCCTGACTCAGAGATTGGGAAGCCCTATATGGTTGGAA GAGAAAGTAAGAGGAGACCCAAAGTATTAGACCACAGAAAGAAGTTCTAA TAGTCAGTGTCAAGAGATTCAGCAGGAGGTTGTGTATCAGGATTTGGGTT TGGGAGTGGTATGGAGCTTACCTATCTCTAAAACGAGCAGGAGGGCAAAA ATGAATCCCAGTCCCAAAGAATTCACTAATGGCCAGCAAACCAACACAGG AACCCCAGCACAGACACACAAGATAGGAAACCAGTTGTTGAAACTACAAT GTAACGGGGCTGATTTAATAAAAACCTGTTACATGAGTTATAGGtttttt ttttttttttttttttttAATGTATGTGCCCCACCTTAGGAAAGCCAGAA ATAATGGCAACGAAGAAATATTCATTCACAGTGAGAAAGCCATTAGAACG TTGGCTGGAACCTAGGGGCATATCGAGGGCCCACGTGGGAAGGACAATGA CAACTTGTTTAGTCCTCACTGGTTTCCCAGTCTGTGGATCTTATTTGAAT hs_chr12_subseq.txt

  18. Human INFG gene

  19. Human INFG gene • From USCS ‘Golden Path website’ genome browser

  20. INFG against ~1/2 Mb region of Chr 12

  21. Assessment • Submit • for either Part 1 or Part 2 the BLAST output, concatenated into one file and annotated • a short summary / discussion of the concepts covered in this practical (< 500 words)

  22. References • Strongly recommend BLAST tutorial on NCBI site • http://www.ncbi.nlm.nih.gov/BLAST/tutorial/ Altschul-1.html • Further “Bioinformatics for quantitative geneticists course notes” J. McEwan • http://www-personal.une.edu.au/~jvanderw/ aabc_materials2004.htm#ModuleC

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