1. Introduction to NCBI & Ensembl tools including BLAST and database searching Incorporating Bioinformatics into the High School Biology Curriculum Fran Lewitter, Ph.D. Founding Director, Bioinformatics & Research Computing Whitehead Institute for Biomedical Research Lewitter (-AT--) iscb.org http://www.iscb.org/iscb-education-home

2. Lewitter-ISCB High School Teachers Workshop – 7/11/14 What I hope you’ll learn What do we learn from database searching and sequence alignments What tools are available at NCBI What tools are available through Ensembl 2

3. First some background info It’s all about alignments. Lewitter-ISCB High School Teachers Workshop – 7/11/143

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5. Doolittle RF, Hunkapiller MW, Hood LE, Devare SG, Robbins KC, Aaronson SA, Antoniades HN. Science 221:275-277, 1983. Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. 5

6. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Why do alignments Use sequence similarity to infer homology and/or structural similarity between 2 or more genes/proteins Identify more conserved regions of a protein, potentially identifying regions of most functional importance Compare and contrast homologs (perhaps into groups) based on shared positions or regions Infer evolutionary distance from sequence dissimilarity 6

7. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Evolutionary Basis of Sequence Alignment Similarity - observable quantity, such as percent identity Homology - conclusion drawn from data that two genes share a common evolutionary history; no metric is associated with this –Paralog – genes related by duplication –Ortholog – genes related by speciation 7

8. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Local vs Global Alignment From Mount, Bioinformatics, 2004, pg 71 GLOBAL LOCAL 8

9. Nucleotide vs Protein If comparing protein coding genes, use protein sequences because of less noise If protein sequences are very similar, it might be more instructive to use DNA sequences Lewitter-ISCB High School Teachers Workshop – 7/11/149

10. Example of simple scoring system for nucleic acids Match = +1 (ex. A-A, T-T, C-C, G-G) Mismatch = -1 (ex. A-T, A-C, etc) Gap opening = - 5 Gap extension = -2 T C A G A C G A G T G T C G G A - - G C T G +1 +1 -1 +1 +1 -5 -2 -1 -1 +1 +1 = -4 10

11. 11Lewitter-ISCB High School Teachers Workshop – 7/11/14 Possible Alignments A:T C A G A C G A G T G B:T C G G A G C T G I.T C A G A C G A G T G T C G G A - - G C T G II.T C A G A C G A G T G T C G G A - G C - T G III.T C A G A C G A G T G T C G G A - G - C T G score=-4 score=-5

12. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Scoring for Protein Alignments - Amino Acid Substitution Matrices PAM - point accepted mutation based on global alignment [evolutionary model] BLOSUM - block substitutions based on local alignments [similarity among conserved sequences] 12

13. Lewitter-ISCB High School Teachers Workshop – 7/11/14 AA Scoring Matrices PAM - point accepted mutation based on global alignment [evolutionary model] BLOSUM - block substitutions based on local alignments [similarity among conserved sequences] Log-odds= pair in homologous proteins pair in unrelated proteins by chance Log-odds= obs freq of aa substitutions freq expected by chance 13

14. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Substitution Matrices BLOSUM 30 BLOSUM 62 BLOSUM 80 % identity PAM 250 (80) PAM 120 (66) PAM 90 (50) % change Increasing similarity 14

15. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Scoring for BLAST Alignments Score = 94.0 bits (230), Expect = 6e-19 Identities = 45/101 (44%), Positives = 54/101 (52%), Gaps = 7/101 (6%) Query: 204 YTGPFCDV----DTKASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQ---AR 256 Y+ FC + + CY G G +YRG T SGA C PW S V Q A+ Sbjct: 198 YSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQ 257 Query: 257 NWGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQCQT 297 GLG H +CRNPD D +PWC VL RL+WEYCD+ C T Sbjct: 258 ALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCST 298 Position 1: Y - Y = 7 Position 2: T - S = 1 Position 3: G - S = 0 Position 4: P - E = -1... Position 9: - - P = -11 Position 10: - - A = -1... Sum 230 Based on BLOSUM62 15

16. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Statistical Significance Raw Scores - score of an alignment equal to the sum of substitution and gap scores. Bit scores - scaled version of an alignment ’ s raw score that accounts for the statistical properties of the scoring system used. E-value - expected number of distinct alignments that would achieve a given score by chance. Lower E-value => more significant. 16

17. Lewitter-ISCB High School Teachers Workshop – 7/11/14 A formula E = Kmn e - S This is the Expected number of high-scoring segment pairs (HSPs) with score at least S for sequences of length m and n. This is the E value for the score S. The parameters K and can be thought of simply as natural scales for the search space size and the scoring system respectively. 17

18. What’s significant? High confidence - >40% identity for long alignments (Rost, 1999 found that sequence alignments unambiguously distinguish between protein pairs of similar and non-similar structure when the pairwise sequence identity >40%) “Twilight zone” – blurry - 20-35% identity “Midnight zone” - <20% identity Lewitter-ISCB High School Teachers Workshop – 7/11/1418

19. Tools of Interest NCBI (US) - http://www.ncbi.nlm.nih.gov/ –BLAST –Entrez EBI/EMBL/WTSI (Europe) –Ensembl (http://www.ensembl.org/) Lewitter-ISCB High School Teachers Workshop – 7/11/1419

20. Lewitter-ISCB High School Teachers Workshop – 7/11/14 NCBI Home Page 20 http://www.ncbi.nlm.nih.gov

21. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Ensembl Home Page 21 http://ensembl.org

22. Hands-On Entrez – finding sequences BLAST – look for similar sequences Ensembl – finding sequences Lewitter-ISCB High School Teachers Workshop – 7/11/1422

23. How is BLAST used? Looking for homologs Identifying domains in proteins Peptide identification Genome annotation Lewitter-ISCB High School Teachers Workshop – 7/11/1423

24. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Remember 1.Don’t be afraid to look at help pages for each website Click, click, click 2.Any analysis tool will give you results 3.Interpret results you find 24

25. Lewitter-ISCB High School Teachers Workshop – 7/11/14 Sequence of Note 1 GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 31 CCCCCTGACG AGCATCACAA AAATCGACGC 61 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ………….. 1371 GTAAAGTCTG GAAACGCGGA AGTCAGCGCC “ Here you see the actual structure of a small fragment of dinosaur DNA, ” Wu said. “ Notice the sequence is made up of four compounds - adenine, guanine, thymine and cytosine. This amount of DNA probably contains instructions to make a single protein - say, a hormone or an enzyme. The full DNA molecule contains three billion of these bases. If we looked at a screen like this once a second, for eight hours a day, it ’ d still take more than two years to look at the entire DNA strand. It ’ s that big. ” (page 103) (Published in 1990) Biotechniques, 1992 25

26. Lewitter-ISCB High School Teachers Workshop – 7/11/14 DinoDNA "Dinosaur DNA" from Crichton's THE LOST WORLD p. 135 GAATTCCGGAAGCGAGCAAGAGATAAGTCCTGGC ATCAGATACAGTTGGAGATAAGGACGACGTGTGG CAGCTCCCGCAGAGGATTCACTGGAAGTGCATTA CCTATCCCATGGGAGCCATGGAGTTCGTGGCGCT GGGGGGGCCGGATGCGGGCTCCCCCACTCCGTTC CCTGATGAAGCCGGAGCCTTCCTGGGGCTGGGGG GGGGCGAGAGGACGGAGGCGGGGGGGCTGCTGGC CTCCTACCCCCCCTCAGGCCGCGTGTCCCTGGTG CCGTGGCAGACACGGGTACTTTGGGGACCCCCCA GTGGGTGCCGCCCGCCACCCAAATGGAGCCCCCC CACTACCTGGAGCTGCTGCAACCCCCCCGGGGCA GCCCCCCCCATCCCTCCTCCGGGCCCCTACTGCC ACTCAGCAGCGCCTGCGGCCTCTACTACAAAC…… Published in 1995 26

27. Lewitter-ISCB High School Teachers Workshop – 7/11/14 >Erythroid transcription factor (NF-E1 DNA-binding protein) Query: 121 MEFVALGGPDAGSPTPFPDEAGAFLGLGGGERTEAGGLLASYPPSGRVSLVPWADTGTLG 300 MEFVALGGPDAGSPTPFPDEAGAFLGLGGGERTEAGGLLASYPPSGRVSLVPWADTGTLG Sbjct: 1 MEFVALGGPDAGSPTPFPDEAGAFLGLGGGERTEAGGLLASYPPSGRVSLVPWADTGTLG 60 Query: 301 TPQWVPPATQMEPPHYLELLQPPRGSPPHPSSGPLLPLSSGPPPCEARECVMARKNCGAT 480 TPQWVPPATQMEPPHYLELLQPPRGSPPHPSSGPLLPLSSGPPPCEARECV NCGAT Sbjct: 61 TPQWVPPATQMEPPHYLELLQPPRGSPPHPSSGPLLPLSSGPPPCEARECV----NCGAT 116 Query: 481 ATPLWRRDGTGHYLCNWASACGLYHRLNGQNRPLIRPKKRLLVSKRAGTVCSHERENCQT 660 ATPLWRRDGTGHYLCN ACGLYHRLNGQNRPLIRPKKRLLVSKRAGTVCS NCQT Sbjct: 117 ATPLWRRDGTGHYLCN---ACGLYHRLNGQNRPLIRPKKRLLVSKRAGTVCS----NCQT 169 Query: 661 STTTLWRRSPMGDPVCNNIHACGLYYKLHQVNRPLTMRKDGIQTRNRKVSSKGKKRRPPG 840 STTTLWRRSPMGDPVCN ACGLYYKLHQVNRPLTMRKDGIQTRNRKVSSKGKKRRPPG Sbjct: 170 STTTLWRRSPMGDPVCN---ACGLYYKLHQVNRPLTMRKDGIQTRNRKVSSKGKKRRPPG 226 Query: 841 GGNPSATAGGGAPMGGGGDPSMPPPPPPPAAAPPQSDALYALGPVVLSGHFLPFGNSGGF 1020 GGNPSATAGGGAPMGGGGDPSMPPPPPPPAAAPPQSDALYALGPVVLSGHFLPFGNSGGF Sbjct: 227 GGNPSATAGGGAPMGGGGDPSMPPPPPPPAAAPPQSDALYALGPVVLSGHFLPFGNSGGF 286 Query: 1021 FGGGAGGYTAPPGLSPQI 1074 FGGGAGGYTAPPGLSPQI Sbjct: 287 FGGGAGGYTAPPGLSPQI 304 27

28. Lewitter-ISCB High School Teachers Workshop – 7/11/14 >Erythroid transcription factor (NF-E1 DNA-binding protein) Query: 121 MEFVALGGPDAGSPTPFPDEAGAFLGLGGGERTEAGGLLASYPPSGRVSLVPWADTGTLG 300 MEFVALGGPDAGSPTPFPDEAGAFLGLGGGERTEAGGLLASYPPSGRVSLVPWADTGTLG Sbjct: 1 MEFVALGGPDAGSPTPFPDEAGAFLGLGGGERTEAGGLLASYPPSGRVSLVPWADTGTLG 60 Query: 301 TPQWVPPATQMEPPHYLELLQPPRGSPPHPSSGPLLPLSSGPPPCEARECVMARKNCGAT 480 TPQWVPPATQMEPPHYLELLQPPRGSPPHPSSGPLLPLSSGPPPCEARECV NCGAT Sbjct: 61 TPQWVPPATQMEPPHYLELLQPPRGSPPHPSSGPLLPLSSGPPPCEARECV----NCGAT 116 Query: 481 ATPLWRRDGTGHYLCNWASACGLYHRLNGQNRPLIRPKKRLLVSKRAGTVCSHERENCQT 660 ATPLWRRDGTGHYLCN ACGLYHRLNGQNRPLIRPKKRLLVSKRAGTVCS NCQT Sbjct: 117 ATPLWRRDGTGHYLCN---ACGLYHRLNGQNRPLIRPKKRLLVSKRAGTVCS----NCQT 169 Query: 661 STTTLWRRSPMGDPVCNNIHACGLYYKLHQVNRPLTMRKDGIQTRNRKVSSKGKKRRPPG 840 STTTLWRRSPMGDPVCN ACGLYYKLHQVNRPLTMRKDGIQTRNRKVSSKGKKRRPPG Sbjct: 170 STTTLWRRSPMGDPVCN---ACGLYYKLHQVNRPLTMRKDGIQTRNRKVSSKGKKRRPPG 226 Query: 841 GGNPSATAGGGAPMGGGGDPSMPPPPPPPAAAPPQSDALYALGPVVLSGHFLPFGNSGGF 1020 GGNPSATAGGGAPMGGGGDPSMPPPPPPPAAAPPQSDALYALGPVVLSGHFLPFGNSGGF Sbjct: 227 GGNPSATAGGGAPMGGGGDPSMPPPPPPPAAAPPQSDALYALGPVVLSGHFLPFGNSGGF 286 Query: 1021 FGGGAGGYTAPPGLSPQI 1074 FGGGAGGYTAPPGLSPQI Sbjct: 287 FGGGAGGYTAPPGLSPQI 304 28