BLAST Tutorial 3 What is BLAST? Basic Local Alignment Search Tool Is a set of similarity search programs designed to explore sequence databases. What are similarity searches good for? One sequence by itself is not informative; it must be analyzed by comparative methods against existing sequence databases to develop hypothesis concerning relatives and function BLAST program Database Query

NameQuery typeDatabase blastnGenomic blastpProtein blastx Translated genomic Protein tblastnProtein Translated genomic tblastx Translated genomic BLAST Databases

Place Query Choose Database ?

BLASTN Databases Gene collection GenBank, EMBL, DDBJ, PDB and NCBI reference sequences (RefSeq) Genomic + Transcript Complete human and mouse genome + transcriptome EST Expressed sequence tags mito Mitochondrial sequences vector Vector subset of GenBank month GenBank, EMBL, DDBJ, PDB from 30 days Envi Environmental samples

Place Query Choose Database Optimize similarity level of the search Threshold for results significance Limit output size Primary word match (16-64 nt) Reward and penalty for matching and mismatching bases Cost to create and extend a gap Remove low information content Limit search to specific organism ?

Search for homologous to chick “olfactory receptor 6” gene

Query sequence Matched Areas of database sequences Global Alignments Local Alignments

Sequence Identifier Sequence description Score(bits) Coverage Identity E value

Score and E value Identities and gaps Strand

Multiple hits on a same subject

Design of the BLAST survey Consider your research question: Are you looking for an particular gene in a particular species?: BLAST against the genome of that species. Are you looking for additional members of a gene family across all species? : BLAST against the gene collection database. Are you looking for exact motif matches? : increase gap penalty or use megablast.

Score and E-value Score (S):  (identities + mismatches)-  gaps Depends on search space Query length(bp) Database length(bp) Depends on scoring system Score Bit Score (S’):

Score and E-value The score is a measure of the similarity of the query to the sequence shown. The E-value is a measure of the reliability of the score. The definition of the E-value is: The probability due to chance, that there is another alignment with a similarity greater than the given S score.

Score and E-value The Size of the E-value The typical threshold for a good E-value from a BLAST search is E=10 -6 ≈e -6 or lower. The reason for such low values is that an E=0.001 in a million entry database would still leave 1000 entries due to chance. An E=e -6 would only leave one entry due to chance.

Given the following parameters: Query length: 150 =1.37 K=0.711 Average Sequence length in database: 270 Number of sequences in database: 4,554,026 Exercise Calculate the S, S’ and E for the following BLAST hit: ACGTCGATCGAGCT |||||||| ||||| AGGTCGTC-GAGGT S = 13-1 = 12 S’= (1.37*12 – ln(0.711))/ln(2) S’= /0.693 S’= 24.2 S:  (Id+MM)-  GP

Exercise Calculate the S, S’ and E for the following BLAST hit: ACGTCGATCGAGCT |||||||| ||||| AGGTCGTC-GAGGT E= 0.711x150x270x4,554,026xe -1.37*12 E= x7.24e-8 E= Given the following parameters: Query length: 150 =1.37 K=0.711 Average Sequence length in database: 270 Number of sequences in database: 4,554,026

Exercise What will be the minimal score in order to achieve a significant E value (e -6 ~10 -6 )? e -1.37S =10 -6 ln ( e -1.37S )=ln(10 -6 ) ln ( )+ln(e -1.37S )= S= S= = /-1.37 S≈ 28.76

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