Sequence Alignments in Biology Workbench

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Sequence Alignments in Biology Workbench

• BIOL/CHEM 4900

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Reading

• Chapter 2 in your textbook
• BLAST help pages on NCBI
• http//www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMDWe
  bPAGE_TYPEBlastDocs

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Types of Alignments

• Pairwise
• Alignment of two sequences
• BLAST, FASTA
• Purpose determine obvious evidence of homology
• Multiple Sequence Alignment
• Alignment of many sequences
• characters and gaps are aligned
• Sequences scored according to similarity and
  number and length of gaps
• CLUSTALW algorithm
• Purpose
• Phylogenetic analysis deeper glance at homology
• Locate conserved regions (primers, functional
  regions, regulatory sites)

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Pairwise Alignments and Databases

• Align characters in two sequences using
  algorithms we have already discussed
• Residueamino acid?protein sequences
• Nucleic acid?DNA and RNA sequences
• Search databases to align one sequence (query)
  with other known sequences (database)
• Determine putative identity of query sequence

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BLAST

• Searches for maximal local alignments
• Inserts gaps to optimize alignment
• Breaks sequence down into words
• Subsequences of fixed length
• NA 16-256 default is 28
• AA 2-3 default is 3
• Searches for those words in database matches and
  scores words (must be a minimal score to be
  considered a hot spot)
• Extends sequence on either side of word and
  scores to look for certain score (threshold
  value)
• Threshold value (or min. score acceptable for a
  hit) is chosen based on the expect value chosen
• Expect expected number of hits that would
  randomly occur without real homology.
• Several types of BLAST algorithms

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Types of BLAST Searches
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FASTA

• A format for sequences
• Also an algorithm for pairwise comparison (like
  BLAST)
• Gapped local alignments using words looks at
  location of words in sequence
• More sensitive than BLAST
• Takes longer to complete
• FASTA protein sequence with protein sequence or
  nucleic sequence with nucleic database
• FASTX, TFASTA, TFASTX translated sequences

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Alignment Assessment

• Results from BLAST or FASTA are not necessarily
  statistically significant
• Look at
• Alignment score (S)
• But, could be aligning by chance
• P score (used in FASTA only)
• Probability that one or more sequences of score
  S would have been found randomly
• E score (used in BLAST only)
• Expected number of sequences that have scores S
  that would be found randomly
• Low values for P and E
• Search result is not random
• Sequences are likely to be related

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BLAST Input

• Follow the link http//www.ncbi.nlm.nih.gov/BLAST
  /Blast.cgi?CMDWebPAGE_TYPEBlastHome

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BLAST Output
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BLAST Output
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BLAST Output
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BLAST in Biology Workbench

• Options
• Database(s) choose up to 16
• E value default 10
• Output
• List of hits with score and E-value
• Alignment diagram
• Length of alignment
• Identities
• Positives (protein)
• Can import sequences of interest

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BLAST on NCBI

• As an alternative, there is a BLAST option on the
  NCBI webpage that uses the same algorithms
• http//www.ncbi.nlm.nih.gov/BLAST/
• Advantages
• More user friendly
• Faster
• Can upload file directly to search page
• Has nr database in nucleotide search (less
  specific)
• Disadvantages
• You cannot automatically import results into
  Biology Workbench for later use
• Only saves for 36 hours

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Lets try it

• Download Nucleic_BLAST_Practice and
  Protein_BLAST_Practice