Computational Biology or Bioinformatics ability to rapidly sequence DNA has led to large databases development of new algorithms data analysis and interpretation.

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Presentation transcript:

Computational Biology or Bioinformatics ability to rapidly sequence DNA has led to large databases development of new algorithms data analysis and interpretation similar concepts also applied to epidemiological databases genetic epidemiology evolutionary genetics align related sequences and search databases

easy to obtain DNA sequence data difficult to predict protein structure and function structure/function can be inferred from sequence similarities similarities identified by aligning DNA or protein sequences alignments can be global or local Sequence Alignments homolog (common ancestor) ortholog (between species) paralog (within species) analog (no common ancestor)

scoring matrix calculates an alignment score eg, match = 0.9 and mismatch = -0.1 for DNA amino acids have different weights (abundance and chemical or structural similarities) BLOSUM and PAM + variants Pair-wise Sequence Alignments Amino Acid Similarities ChemicalPhysical A, G D, E F, Y K, R I, L, M, V Q, N S, T C, S D, L, N E, Q F, H, W, Y I, T, V K, M, R GCGCCTC ||| || GCGGGTC (5 x 0.9) + (2 x -0.1) = 4.3

Multiple sequence alignments gives 1 st approximation of best score human eye + biological insight better at refining the alignments gap penalties (opening and extending) optimal penalties depend on relatedness of sequences 'trial and error' approach alignment with maximum score is returned for prescribed gap penalties and scoring matrix not necessarily most biological significant Pair-wise Sequence Alignments

two types: 1 o (original biological data) 2 o (value added) three 1 o DNA databases GenBank EMBL DDBJ subdivisions (taxonomic groups, genome projects, ESTs, etc) annotated to include ancillary information (author, publications, etc.) Databases

Searching Databases text-based (annotations) gene name, authors, species, etc. information retrieval systems Entrez can access all databases + medline sequence comparisons submit query sequence compare to all sequences in database(s) pairwise is too time consuming heurisitic programs (eg, FASTA and BLAST ) match short sequence fragments alignments of sequence regions showing promise scores and statistics

Basic Local Alignment Search Tool

Doing a BLAST Search BLAST/ choose BLAST program paste in query sequence or acc. no. BLAST! change default options: database (nr = non-redundant) scoring matrix and gap penalties filtering E-value cutoff (ie, Expect) limit subset of database (organism, keyword, etc.) display options (eg, # of descriptions, alignments, etc.)

Blast Search Results Query= Pbpp58b (423 letters) Database: nr (493,611 sequences; 154,780,071 total letters) Score E Sequences producing significant alignments: (bits) Value sp|Q08168|HRP_PLABE 58 KD PHOSPHOPROTEIN (HEAT SHOCK-RELATED PRO e-90 gb|AAC | (L21710) 58 kDa phosphoprotein [Plasmodium berghei] 329 3e-89 pir||T10455 heat shock related protein - Plasmodium berghei >gi| e-65 sp|P50503|HIP_RAT HSC70-INTERACTING PROTEIN >gi| |emb|CAA e-22 sp|P50502|HIP_HUMAN HSC70-INTERACTING PROTEIN (PROGESTERONE RECE e-16 gb|AAF | (AE003429) CG2947 gene product [Drosophila melano e-16 pir||T24865 hypothetical protein T12D8.8 - Caenorhabditis elegan e-16 pir||T04562 hypothetical protein T12H Arabidopsis thalian e-14. emb|CAA | (X89416) protein phosphatase 5 [Homo sapiens] pdb|1A17| Tetratricopeptide Repeats Of Protein Phosphatase ref|NP_ || protein phosphatase 5, catalytic subunit >gi| pir||S52570 phosphoprotein phosphatase (EC ) 5, catalyti Probabilitydatabase | accession # | entry name or locus

Example of Blast Alignment >pir||T24865 hypothetical protein T12D8.8 -Caenorhabditis elegans (Length = 422) Score = 86.2 bits (210), Expect = 5e-16 Identities = 44/101 (43%), Positives = 60/101 (58%), Gaps = 2/101 (1%) Query: 119 EAVDLVENKKYEEALEKYNKIISFGNPSAMIYTKRASILLNLKRPKACIRDCTEALNLNV 178 +A + N ++ AL + I SAM++ KRA++LL LKRP A I DC +A+++N Sbjct: 121 KAQEAFSNGDFDTALTHFTAAIEANPGSAMLHAKRANVLLKLKRPVAAIADCDKAISINP 180 Query: 179 DSANAYKIRAKAYRYLGKWEFAHADMEQGQKIDYDE--NLW 217 DSA YK R +A R LGKW A D+ K+DYDE N W Sbjct: 181 DSAQGYKFRGRANRLLGKWVEAKTDLATACKLDYDEAANEW 221 Score = 41.4 bits (95), Expect = Identities = 16/34 (47%), Positives = 23/34 (67%) Query: 9 LKKFVASCEENPSILLKPELSFFKDFIESFGGKI 42 LK+FV C+ NP++L PE FFKD++ S G + Sbjct: 7 LKQFVGMCQANPAVLHAPEFGFFKDYLVSLGATL 40 Gap Matches A 2 nd high scoring segment

Blast Search Results Query= Pbpp58b (423 letters) Database: nr (493,611 sequences; 154,780,071 total letters) Score E Sequences producing significant alignments: (bits) Value sp|Q08168|HRP_PLABE 58 KD PHOSPHOPROTEIN (HEAT SHOCK-RELATED PRO e-90 gb|AAC | (L21710) 58 kDa phosphoprotein [Plasmodium berghei] 329 3e-89 pir||T10455 heat shock related protein - Plasmodium berghei >gi| e-65 sp|P50503|HIP_RAT HSC70-INTERACTING PROTEIN >gi| |emb|CAA e-22 sp|P50502|HIP_HUMAN HSC70-INTERACTING PROTEIN (PROGESTERONE RECE e-16 gb|AAF | (AE003429) CG2947 gene product [Drosophila melano e-16 pir||T24865 hypothetical protein T12D8.8 - Caenorhabditis elegan e-16 pir||T04562 hypothetical protein T12H Arabidopsis thalian e-14. emb|CAA | (X89416) protein phosphatase 5 [Homo sapiens] pdb|1A17| Tetratricopeptide Repeats Of Protein Phosphatase ref|NP_ || protein phosphatase 5, catalytic subunit >gi| pir||S52570 phosphoprotein phosphatase (EC ) 5, catalyti Probabilitydatabase | accession # | entry name or locus

Example of Blast Alignment >sp|P50503|HIP_RAT HSC70-INTERACTING PROTEIN >gi| |emb|CAA | (X82021) Hsc70-interacting protein [Rattus norvegicus] (Length = 368) Score = 106 bits (261), Expect = 5e-22 Identities = 60/224 (26%), Positives = 97/224 (42%) Query: 1 MDIEKIEDLKKFVASCEENPSILLKPELSFFKDFIESFGGKIKKDKMGYXXXXXXXXXXX 60 MD K+ +L+ FV C ++PS+L E+ F ++++ES GGK+ Sbjct: 1 MDPRKVSELRAFVKMCRQDPSVLHTEEMRFLREWVESMGGKVPPATHKAKSEENTKEEKR 60 (SDEEEEDEEEEEEEEEDDDPEKLE) Query: 61 XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXAVECPPLAPXXXXXXXXXXXXXXCKLKEEA P + K A Sbjct: 61 DKTTEDNIKTEEPSSEESDLEIDNEGVIEADTDAPQEMGDENAEITEAMMDEANEKKGAA 120 Query: 121 VDLVENKKYEEALEKYNKIISFGNPSAMIYTKRASILLNLKRPKACIRDCTEALNLNVDS 180 +D A++ + I A++Y KRAS+ + L++P A IRDC A+ +N DS Sbjct: 121 IDALNDGELQKAIDLFTDAIKLNPRLAILYAKRASVFVKLQKPNAAIRDCDRAIEINPDS 180 Query: 181 ANAYKIRAKAYRYLGKWEFAHADMEQGQKIDYDENLWDMQKLIQ 224 A YK R KA+R LG WE A D+ K+DYDE+ M + +Q Sbjct: 181 AQPYKWRGKAHRLLGHWEEAARDLALACKLDYDEDASAMLREVQ 224 Filtering

LOCUS RNHSRP 1694 bp mRNA ROD 14-JAN-1996 DEFINITION R.norvegicus mRNA for heat shock related protein. ACCESSION X82021 REFERENCE 2 (bases 1 to 1694) AUTHORS Hohfeld,J., Minami,Y. and Hartl,F.U. JOURNAL Cell 83 (4), (1995) MEDLINE FEATURES Location/Qualifiers source /organism="Rattus norvegicus" gene /gene="hip" CDS /product="Hsc70-interacting protein" /protein_id="CAA " /db_xref="SWISS-PROT:P50503" /translation="MDPRKVSELRAFVKMCRQDPSVLHTEEMRFLREWVESMGGK QDVAQNPSNMSKYQNNPKVMNLISKLSAKFGGHS" BASE COUNT 542 a 342 c 423 g 387 t 1 gcgtcgacgg gcttggcatc gggcctccgc agccgcccac cgccagaagc ttccagcctc aaaaaaaaaa aaaa //

| Acidic Domain Pbe MDIEKIEDLKKFVASCEENPSILLKPELSFFKDFIESFGGKIKKD KMGYEKMKSEDSTEEKSDEEEEDEEEEEEEEEDD 79 Rat MDPRKVSELRAFVKMCRQDPSVLHTEEMRFLREWVESMGGKVPPATHKAKSEENTKEEKRDKT-TEDNIKTEEPSSEESD 79 ** *...*. ** *..**.*. *..*.....**.*** *.....*..**.* | | TPR || Pbe DPEKLELIKEEAVECPPLAPIIEGELSEEQIEEICKLKEEAVDLVENKKYEEALEKYNKIISFGNPSAMIYTKRASILLN 159 Rat LEIDNEGVIEADTDAPQEMGDENAETTEAMMDEANEKKGAAIDALNDGELQKAIDLFTDAIKLNPRLAILYAKRASVFVK 159. *. *... *...*.*..... *.* *..... *.... *..*.****.... TPR || TPR | | Basic Domain Pbe LKRPKACIRDCTEALNLNVDSANAYKIRAKAYRYLGKWEFAHADMEQGQKIDYDENLWDMQKLIQEKYKKIYEKRRYKIN 239 Rat LQKPNAAIRDCDRAIEINPDSAQPYKWRGKAHRLLGHWEEAARDLALACKLDYDEDASAMLREVQPRAQKIAEHRRKYER 239 *..*.* ****. *...* ***..** *.**.* **.** * *... *.****..*..*..** *.**. | || Pbe KEEEKQRLKREKELKKKLAAKKKAEKMYKENNKRENYDSDSSDSSYSEPDFSGDFPGGMPGGMPGMPGGMGGMGGMPGMP 319 Rat KREEREIKERIERVKKAREEHEKAQRE EEARRQSGSQFGSFPGGFPGGMPGNFPGGMPGMGG 301 * **...*..**....** *..*.*.*******..** **** GGMP Repeat Domain | Pbe GGFPGMPGGMPGGMPGGMGGMPGMPGGMPGGMGGMPGMPGGMPDLNSPEMKELFNNPQFFQMMQNMMSNPDLINKYASDP 399 Rat AMP GMAGMPGLNEILSDPEVLAAMQDPEVMVAFQDVAQNPSNMSKYQNNP 351.** **.**** *..**.....*...*.. **...**...* Pbe KYKNIFENLKNSDLGGMMGEKPKP 423 Rat KVMNLISKLSAKFGG HS 368 *.*....*... *.