Introduction to Bioinformatics - Tutorial no. 2

BLAST

BLAST

BLAST – Outline

Sequence Alignment Complexity and indexing BLASTN and BLASTP

Basic parameters PAM and BLOSUM matrices Affine gap model E Values (once again)

Advanced BLAST

Databases BLAST options BLAST output Taxonomic BLAST Pairwise BLAST

Name Query type Database

blastn Genomic Genomic

blastp Protein Protein

blastx Translated genomic Protein

tblastn Protein Translated genomic

tblastx Translated genomic Translated genomic

Genomic translations test all 6 possibilities:

3x for codon frames, 2x for reverse complement

BLAST Variations

BLASTN Databases

nrGenBank, EMBL, DDBJ, PDB and NCBI

reference sequences (RefSeq)

htgs High-throughput genomic sequences (draft)

pat Patented nucleotide sequences

mito Mitochondrial sequences

vector Vector subset of GenBank

month GenBank, EMBL, DDBJ, PDB from 30 days

chrom Contigs and chromosomes from RefSeq

BLASTP Databases

nrGenBank CDS translations, RefSeq,

PDB, SWISS-PROT, PIR, PRF

swissprot SWISS-PROT

pat Patented protein sequences

pdb Protein Data Bank

monthGenBank CDS translations, PDB,

SWISS-PROT, PIR, PRF from 30 days

BLASTN/P Options (1)

Only search part of database using NCBI Entrez query format

Search specific

organism

Remove low information content, e.g. short repeats or

rich in only 2 nucleotides

Remove known human repeats

(LINEs, SINEs)

BLASTN/P Options (2)Threshold for results

significance

Use index based on words of 7, 11 or 15

nucleotides Costs to open and extend gap, score for nucleotide

match or mismatch. Allowed gap scores: 10/1, 10/2, 11/1, 8/2, 9/2

BLASTP Options

Scoring matrix: PAM, etc…

Search for a motif (PSI-BLAST)

Costs to open and extend gap

BLASTN/P Formatting (1)

Show colored bar chart

Number of sequences listed

Number of alignments shown

Other (less important) options on

what to show

BLASTN/P Formatting (2)

How to display alignments

Only show results which match Entrez search or are from specific organism

Only show results with E values in this range

BLASTN Results

Query sequence representation

Matched areas of database sequences

BLAST Output Header

Request ID for later retrieval

Query sequence details

Database details

Tax BLAST

BLAST Alignments (1)

Sequence Identifier

Sequence description

Score andE value

BLAST Alignments (2)

Normalized score of alignment

Expected number of such hits (2e-11 = 2 10-11)

Number of exact matches

Number of matches with positive score

Number of insertion / deletions

BLAST Alignments (3)

Query sequenceExact matchInsertion / deletion

Matched sequence

Mismatch with positive

score

Position within sequence Masked low complexity region

Expectation Values

Increases linearly with

length of query sequence

Increases linearly with

length of database

Decreases exponentially with score of

alignment

Tax BLAST

Lineage of organism with strongest hit

Score of organism’s strongest hit

Number of organism hits

Shared ancestry in taxonomic tree

BLAST2SEQ

Scoring scheme

Type of program

Gap model,Expect Value,

Advanced options

Sequences

Scoring matrix

SequencesGO !

This tool produces the alignment of two given sequences using BLAST engine for local alignment.

QuestionsYou have two query sequences: query1 and query2:

>query1CCGTCCGTCCGTCGTCCTCCTCGCTTGCGGGGCGCCGGGCCCGTCCTCGAGCCCCCNNNNNCCGTCCGGCCGCGTCGGGGCCTCGCCGCGCTCTACCTACCTACCTGGTTGATCCTGCCAGTAGCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTACGCACGGCCGGTACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATGGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCCCTCTCCGGCCCCGGCCGGGGGGCGGGCCGCGGCGGCTTTGGTGACTCTAGATAACCTCGGGCCGATCGCACGCCCCCCGTGGCGGCGACGACCCATTCGAACGTCTGCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTACCATGGTGACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCCCGACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTCCACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATCTTGGGAGCGGGCGGGCGGTCCGCCGCGAGGCGAGCCACCGCCCGTCCCCGCCCCTTGCCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGTCCCGCGGGGCCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATACCGCAGCTAGGAATAATGGAATAGGACCGCGGTTCTATTTTGTTGGTTTTCGGAACTGAGGCCATGATTAAGAGGGACGGCCGGGGGCATTCGTATTGCGCCGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGACCAGAGCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATAAACGATGCCGACCGGCGATGCGGCGGCGTTATTCCCATGACCCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGACAGGATTGACAGATTGATAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGAGACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCCAACTTCTTAGAGGGACAAGTGGCGTTCAGCCACCCGAGATTGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCTACACTGACTGGCTCAGCGTGTGCCTACCCTACGCCGGCAGGCGCGGGTAACCCGTTGAACCCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAATTCCCAGTAAGTGCGGGTCATAAGCTTGCGTTGATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGGATGGTTTAGTGAGGCCCTCGGATCGGCCCCGCCGGGGTCGGCCCACGGCCTGGCGGAGCGCTGAGAAGACGGTCGAA

Questions>query2TACGAACGCTGGCGGCATGCTAATACATGCAAGTCGAACGAGACCTTCGGGTCTAGTGGCGCACGGGTGGCTAACGCGTGGGAATCTGCCCTTGGGTTCGGAATAACTTCGGGAAACTGAAGCTAATACCGGATGATGACGAAAGTCCAAAGATTTATCGCCCAGGGATGAGCCCGCGTAGGATTAGCTAGTTGGTGGGGTAAAGGCTCACCAAGGCAACGATCCTTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTTACCCGAGATGATAATGACAGTATCGGGAGAATAAGCTCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGAGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGCGATTTAAGTCAGAGGTGAAAGCCCGGGCTCAACCCCGAACTGCCTTTGAGACTGGATTGCTAGAATCTTGGAGAGGCGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGCGAAGGCGGCTCGCTGGACAAGTATTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATAACTAGCTGCCGGGGCACATGGTGTTTCGGTGGCGCACGTAACGCATTAAGTTATCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCTGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCGTTTGACATCCTCATCGCGGATTTCAGAGATGATTTCCTTCAGTTCGGCTGGATGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTGCCAGCATTTAGTTGGGTACTCTAAAGGAACCGCCGGTGATAAGCCGGAGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGCGCTGGGCTACACACGTGCTACAATGGCGACTACAGTGGGCTGCAACCGTGCGAGCGGTAGCTAATCTCCAAAAGTCGTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGGCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCGCCCGTCACACCATGGGATTTGGATTCACCCGAAGGCACTGCGTTAACCCGCAAGGGAGACAGGTGACCACGGTGGGTTTAGAGACTGGGGTGAA

QuestionsUsing BLASTN • Find what do each one of these sequences code

for.

Questions

Questions

• To which organism each sequence is related? • Do these sequences code for proteins?

Pretend the information for answering previous questions is not available to you could you suggest a way to answer these questions anyway?

BLASTX

Questions

• Look carefully at the e-value column of the first 50 results of each query. What can you learn about these sequences? Are these sequences generally conserved between other organisms?

5 last answers

Questions

• Use bl2seq to align the two query sequences. What can you say about the relation between them? Based does this last result make sense?

QuestionsYou have two query sequences.>query3 ATGTCTGCTCCACAAGCCAAGATTTTGTCTCAAGCTCCAACTGAATTGGAATTACAAGTTGCTCAAGCTTTCGTTGAATTGGAAAATTCTTCTCCAGAATTGAAAGCTGAGTTGAGACCTTTGCAATTCAAGTCCATCAGAGAAGT

>query4 GTATGTTATTAATTTGAATCTAAACTTAAGAATAATGGAGAGTAACAAAGGAAAAAAGTGTGAACGGGACGATACCAGAATGTTTCAATCTAGAAAAGTATAAAAGATAAGGACTAGGACTCAAATGTATTTGGCTGACTATCGCCTGAACCTTGATGCTAAGCAAATACCATATCTTCAAGAAAAAGCCTACTCCAGTGTTTAAGAAGAAGGGAACGATTTACTAGATCATGCTATACGCAGTAAGGTTCTGATAGTTAATTACAATCGGTCCAAGTTCTAAGCGGTGTCGTCCATGCATATATCATTTACAAGTTACTGGCGTCAACTCTTCAAATATTCAAAATATCACCTAATCAAACTTACTAACATTTTCCTTTTTTGTTTTCCTTCTTTTATAG

Now use BlastX

• To what protein does these sequences code for?

• are these proteins conserved in other organisms?

QuestionsNow use BlastX

• To what protein does these sequences code for?

• are these proteins conserved in other organisms?

A conserved protein component of the small (40S) subunit of S. cerevisiae.

Query 3Query 4

No protein – e-value 3.2

Questions• You are told that the sequences were extracted from the

same gene. How could you explain the above results?

• Answer: query4 is extracted from a non-coding region (intron) and thus doesn’t code for any protein.