1. Workshop OUTLINE Part 1: Introduction and motivation How does BLAST work? Part 2: BLAST programs Sequence databases Work Steps Extract and analyze results

2. BLAST programs 2 All types of searches are possible Query:DNAProtein Database:DNAProtein blastn – nuc vs. nuc blastp – prot vs. prot blastx – translated query vs. protein database tblastn – protein vs. translated nuc. DB tblastx – translated query vs. translated database

3. Amino acid sequence – most suitable for homology search The database and the query can be either nucleotides or amino acids! We prefer amino acid sequence: -amino acid sequence is more conserved -20 letter alphabet. Two random hits share 5% identity in average (comparing to 25% in DNA seq). -protein comparison matrices are more sensitive. - protein databases are smaller – less random hits. - we want to conclude about the structure- proteins are much more relevant. BLAST programs

4. Where? (to find homologues) Structural templates- search against the PDB Sequence homologues- search against SwissProt or Uniprot (recommended!) How many? As many as possible, as long as the MSA looks good (next week…) General Issues

5. How long? (length of homologues) Fragments- short homologues (less than 50,60% the query’s length) = bad alignment Ensure your sequences exhibit the wanted domain(s) N/C terminal tend to vary in length between homologues How close? (distance from query sequence) All too close- no information Too many too far- bad alignment Ensure that you have a balanced collection! General Issues

6. From who? (which species the sequence belongs to) Don’t care, all homologues are welcome Orthologues/paralogues may be helpful Sequences from distant/close species provide different types of information Which method? (BLAST/PSI-BLAST) Depends on the protein, available homologues, the goal in mind… General Issues

7. Sequence databases Where do we want to search? DNA sequences ESTs- no annotated coding sequence pool. the largest pool of sequence data for many organisms (NCBI) NR- All GenBank + EMBL + DDBJ + PDB sequences. No longer "non- redundant" due to computational cost. Genomes a specific organisms RefSeq- mRna or genomic- an annotated collection from NCBI Reference Sequence Project. EMBL- Europe's primary nucleotide sequence resource (EBI) ….

8. Sequence databases Where do we want to search? Protein databases: PDB- the sequences of proteins for which structures are available NR (non-redundant)- Non-redundant GenBank CDS translations + PDB + SwissProt + PIR + PRF, excluding those in env_nr RefSeq- sequences from NCBI Reference Sequence project.NCBI Reference Sequence project Proteins of a specific organisms Uniprot –swissprot or trembl ….

9. Sequence databases Where do we want to search? UniProt UniProt is a collaboration between the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). European Bioinformatics Institute (EBI) Swiss Institute of Bioinformatics (SIB)Protein Information Resource (PIR) In 2002, the three institutes decided to pool their resources and expertise and formed the UniProt Consortium.

10. Sequence databases Where do we want to search? UniProt The world's most comprehensive catalog of information on proteins- Sequence, function & more… Comprised mainly of the databases: – SwissProt – 366226 last year, 412525 protein entries now – high quality annotation, non-redundant & cross-referenced to many other databases. – TrEMBL - 5708298 last year, 7341751 protein entries now – computer translation of the genetic information from the EMBL Nucleotide Sequence Database  many proteins are poorly annotated since only automatic annotation is generated

11. Overall work steps 1.Run the search- 1.Select database 2.E-value threshold 3.BLAST or PSI-BLAST- how many rounds? 2.Take out sequences 1.HSP or full sequences 2.Can (should!) filter out redundant and sequences that are too short (fragments) 3.Usually- align sequences- choose alignment program 4.View alignment with BioEdi tor another program 5.Calculate trees, conservatino scores (conseq) etc…

12. Multiple Sequence Alignment (MSA) Overall work steps Perform alignment of a large collection of sequences Many algorithms, leading ones: 1.ClustalW 2.MUSCLE 3.T-COFFEE

13. Examining BaliBase 2005… Edgar, R.C., 2004 MUSCLE is superior! Overall work steps

14. BLAST NCBI

15. All program types Many databases to chose from, both nucleotide and protein 12 genome-specific databases Can also look for conserved domain, SNPs and more… The well-known server http://blast.ncbi.nlm.nih.gov/Blast.cgi

16. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi BLASTp BLAST NCBI

17. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi Query Sequence Database Run BLAST NCBI BLASTp

18. As many as possible Matrix BLAST NCBI Evalue

19. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi Mark all Mark only wanted BLAST NCBI

20. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi BLAST NCBI

21. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi BLAST NCBI

22. BLAST EBI

23. http://www.ebi.ac.uk/blastall/index.html Many databases, including UniProt Insert sequence RUN Get maximum number of alignments! BLAST EBI

24. http://www.ebi.ac.uk/blastall/index.html Send sequences to ClustalW Mark all or wanted Get sequences BLAST EBI

25. PSI-BLAST

26. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi Query Sequence Database Run PSI-BLAST NCBI PSI-BLAST

27. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi Pre-calculated PSSM PSI-BLAST NCBI Threshold for inclusion in PSSM

28. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi PSI-BLAST NCBI Run next round Include sequence in the PSSM Not found in previous round

29. http://www.ebi.ac.uk/blastpgp/ Query Sequence Database Run PSI-BLAST EBI Number of iterations

30. (PSI-)BLAST on ConSeq, extract sequence & align

31. PSI-BLAST on ConSeq The ConSeq webserver Calculates evolutionary conservation scores that are than displayed on the sequence. Requires a Multiple Sequence Alignment (MSA)- if nor provided, can create one automatically Runs (PSI-)BLAST, extracts hits from the BLAST results, filters according to e-value and aligns the sequences.

32. PSI-BLAST on ConSeq The ConSeq webserver-http://conseq.tau.ac.il/http://conseq.tau.ac.il/

33. PSI-BLAST on ConSeq The ConSeq webserver-http://conseq.tau.ac.il/http://conseq.tau.ac.il/ Query sequence Email

34. PSI-BLAST on ConSeq The ConSeq webserver-http://conseq.tau.ac.il/http://conseq.tau.ac.il/ Alignment algorithm Database- swissprot or uniprot No. of homologues Iterations E-value

35. PSI-BLAST on ConSeq The ConSeq webserver-http://conseq.tau.ac.il/http://conseq.tau.ac.il/

36. PSI-BLAST on ConSeq The ConSeq webserver-http://conseq.tau.ac.il/http://conseq.tau.ac.il/ All BLAST hits MSA

37. Summary of web servers: 1. PSI-BLAST at NCBI- -Can control PSSM, included sequences & threshold -All types of BLAST programs -Not against UniProt- SwissProt or NR -Against RefSeq and NT -Full sequences downloaded like BLAST -Number of sequences up to 2000 NCBI vs. EBI vs. ConSeq

38. Summary of web servers: 2. BLAST at EBI – - Against UniProt or EMBL, not NR or specific genomes - Can’t control PSSM- just get last round - Download and align only full sequences - The number of presented sequences is limited to 500 - blastN, blastP, tblastN, tblastX NCBI vs. EBI vs. ConSeq

39. Summary of web servers: 3. BLAST at ConSeq – Get HSPs, not entire sequences!!! Only blastP Search uniprot/swissprot Still, can’t control all options… such as redundancy and minimal length of HSP NCBI vs. EBI vs. ConSeq

40. (PSI-)BLAST via Max-Planck

41. Run (PSI-) BLAST Send HSP or full sequences to an alignment program Forward HSP to filtration via “BLAMMER” Download filtered sequences Align the sequences via program of choice

42. (PSI-)BLAST via Max-Planck BLAST at Max-Planc http://toolkit.tuebingen.mpg.de/sections/search Databases- swissprot, tremble, NR, env, pdb or any combination for proteins, but only NT for DNA. All BLAST programs Main advantage- you can easily extract and filter the HSPs, on top of full sequences.

43. The Query Protein Name: Dihydrodipicolinate reductase Enzyme reaction: Molecular process: Lysine biosynthesis (early stages) Organism: E. coli Sequence length: 273 aa

44. Query: DAPB_ECOLI <DAPB_ECOLI MHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAGELAGAG KTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMVIGTTGFDEAGKQ AIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDYTDIEIIEAHHRHKVDAPSGTA LAMGEAIAHALDKDLKDCAVYSREGHTGERVPGTIGFATVRAGDIVGEHTAMFADIGE RLEITHKASSRMTFANGAVRSALWLSGKESGLFDMRDVLDLNNL The Query Protein

45. (PSI-)BLAST via Max-Planck http://toolkit.tuebingen.mpg.de/psi_blast/ Choose database or databases (selecting a few using CTRL) Upload sequence or MSA

46. (PSI-)BLAST via Max-Planc Save PSi-BLAST result

47. (PSI-)BLAST via Max-Planck E-value threshold can be assessed using the distribution

48. Filter Results via Max-Planck Forward results to BLAMMER

49. BLAMMER Suppose to create MSAs from BLAST results, we will use it just to filter the results and then align them via MUSCLE or another known MSA program. Filter according to: E-value Min. coverage- min. percent of the query protein Max. redundancy- extract similar sequences Max. number of homolgoues- if wanted Filter Results via Max-Planck http://toolkit.tuebingen.mpg.de/blammer/

50. Filter Results via Max-Planck http://toolkit.tuebingen.mpg.de/blammer Forwarded PSI- BLAST result Filtering parameters

51. Filter Results via Max-Planck Save & then re-align!

52. Align the BLAST sequences

53. Align via Max-Planck http://toolkit.tuebingen.mpg.de/sections/alignment

54. 1.Forward BLAST to MUSCLE, MAFFT etc... Choose program Use hits or full sequences Align via Max-Planck

55. 2. Filter via BLAMMER and then ALIGN: Upload the results of the BLAMMER – downloaded file

56. Align via Max-Planck Alignment results: Save the alignment

57. Alignmen viewing & editing BioEdit http://www.mbio.ncsu.edu/BioEdit/BioEdit.html Easy-to-use sequence alignment editor View and manipulate alignments up to 20,000 sequences. F our modes of manual alignment: select and slide, dynamic grab and drag, gap insert and delete by mouse click, and on-screen typing which behaves like a text editor. Reads and writes Genbank, Fasta, Phylip 3.2, Phylip 4, and NBRF/PIR formats. Also reads GCG and Clustal formats

58. Easiest Using Bioedit http://www.mbio.ncsu.edu/BioEdit/bioedit.html Alignmen viewing & editing

59. Easiest Using Bioedit http://www.mbio.ncsu.edu/BioEdit/bioedit.html Find a specific sequence: “Edit-> search -> in titles” Erase\add sequences: “Edit-> cut\paste\delete sequence” “Sequence Identity matrix” under “Alignment”- useful for a rough evaluation of distances within the alignment. After taking out sequences, “Minimize Alignment” under “Alignment” takes out unessential gaps. Can save an image using: “File -> Graphic View” & then “Edit -> Copy page as BITMAP” Alignmen viewing & editing

60. Each sequence is a different story  adjust parameters: BLAST- E-value, substitution matrix, gap penalties, database, minimum length, redundancy level, fragment overlap… PSI-BLAST- BLAST parameters + PSSM inclusion threshold (or chose manually), number of rounds… Try using HSP or full sequences, different MSA programs… No “Miracle solution” 

61. THANKS Some slides were taken from previous presentations by members of the Pupko lab and Prof. Beni Chor