1. Basic Introduction of BLAST Jundi Wang School of Computing CSC691 09/08/2013

2. 2 Overview 1.Introduction of BLAST  Background of BLAST  Programs in BLAST  Function of BLAST 2.Application of BLAST  BLAST web version  Stand-alone BLAST

3. Background of BLAST  BLAST (Basic Local Alignment Search Tool): 1.The most widely used sequence similarity tool. 2.BLAST is a family of programs: a) Compare protein queries to protein databases b) Compare nucleotide queries to nucleotide databases 3

4. Background of BLAST  The Mechanism of BLAST Finding similar sequences: BLAST finds similar sequences by locating short matches between the two sequence. After the first match, BLAST begins to make local alignments. 4

5. Programs in BLAST There are some different BLAST programs available for different analytic purposes.  Nucleotide-nucleotide BLAST (blastn) This program, given a DNA query, returns the most similar DNA sequences from the DNA database that the user specifies.  Protein-protein BLAST (blastp) This program, given a protein query, returns the most similar protein sequences from the protein database that the user specifies. 5

6. Programs in BLAST  Nucleotide 6-frame translation-protein (blastx) This program compares the six-frame conceptual translation products of a nucleotide query sequence against a protein sequence database.  Nucleotide 6-frame translation-nucleotide 6-frame translation (tblastx) This program translates the query nucleotide sequence in all six possible frames and compares it against the six-frame translations of a nucleotide sequence database.  Protein-nucleotide 6-frame translation (tblastn) 6

7. Programs in BLAST  Protein-nucleotide 6-frame translation (tblastn) This program compares a protein query against the all six reading frames of a nucleotide sequence database. 7

8. Six-Frame Translation Once a gene has been sequenced it is important to determine the correct open reading frame (ORF). Every region of DNA has six possible reading frames, three in each strand. The ORF that is used determines which amino acids will be encoded by a gene. Typically only one reading frame is used in translating a gene (in eukaryotes). The ORF starts with an start codon (ATG) and ends with a stop codon (TAA, TAG, or TGA). 8

9. Six-Frame Translation Example: 9

10. Function of BLAST  BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. 10

11. Application of BLAST  BLAST web version: Advantage: 1.It is convenient to operate. 2.Synchronously updates the databases. Weakness: 1.It is not good enough to analyze large-scale data. 2.Programmer cannot customize the database. http://www.ncbi.nlm.nih.gov/BLAST/ 11

12. Application of BLAST  Stand-alone BLAST: Advantage: 1.It can be used to analyze large-scale data. 2.Programmer can customize the database. 3.Programmer can download different version for different operating system. Weakness: 1.It is difficult to user who don’t have computer science background. ftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST/ 12

13. Application of BLAST  Statistics in BLAST 1.Score: It is a value calculated from the number of gaps and substitutions associated with each aligned. 2. E value: It describes the likelihood that a sequence with a similar score will occur in the database by chance. 13

14. Application of BLAST 3. Identities: It describes the identity between query sequence and the sequence from database. 4.Positive: It describes the similarity between query sequence and the sequence from database. 5.Gaps: It describes the gaps between query sequence and the sequence from database. 14

15. 15 Application of BLAST (web version) NCBI BLAST web page Nucleotide Alignment Protein Alignment

16. 16 Application of BLAST (web version) Query Sequence Upload File Query Subrange Select Database

17. 17 Application of BLAST (web version) Select Algorithm E value limitation

18. 18 Application of BLAST (web version) Click “Mouse” to check the detail

19. Application of BLAST (web version) 19 100% Identity No Gap The Value of score is the result of Score Matrix

20. Application of BLAST (web version) 20 All compared sequence NCBI Accession ID

21. Application of BLAST (Stand-alone Version)  Download and install Stand-alone BLAST ftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST/  Download the database from NCBI ftp://ftp.ncbi.nlm.nih.gov/blast/db/  Download and install Activeperl from ActiveState http://www.activestate.com/activeperl 21

22. Application of BLAST (Stand-alone Version)  Build local database 1.Enter the BLAST folder and create a database (db) folder. 2.Extract the downloaded database into the db folder.  Link the database to the BLAST 1.Execute cmd.exe and link the database to the BLAST by Perl.  Modify the environment variables 1. Set the new path variable in order to make the BLAST to be recognized. 22

23. Application of BLAST (Stand-alone Version)  Create a query sequence with a FASTA format. 23 Start with “>” Follow by the name or description of the query sequence

24. Application of BLAST (Stand-alone Version) Example: Compare the query sequence with the sequence from the “refseq_rna.00” database. 24 Different program in BLAST package Link the “refseq_rna.00” to the BLAST Name of database

25. Application of BLAST (Stand-alone Version) 25 The basic information of the current database

26. Application of BLAST (Stand-alone Version) 26 Execute “blastn” program Import the query sequence Import the target database Report the result in a new file

27. Application of BLAST (Stand-alone Version) 27 The length of compared sequence NCBI Accession ID All compared sequenceStatistic evaluation

28. Application of BLAST (Stand-alone Version) 28

29. Application of BLAST (Stand-alone Version) 29

30. Summary DNA Sequencing in a new species NCBI BLAST Database Query Import Output

31. Thank You 31