BIO341 Gene Discovery Section 4 Bioinformatics and genome analysis

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BIO341Gene DiscoverySection 4Bioinformatics
and genome analysis

• Jasper Rees
• Department of Biochemistry, UWC
• www.biotechnology.uwc.ac.za/teaching/BIO341

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Bioinformatics and genome analysis

• Bioinformatics - the analysis of biological
  information usually applied to molecular data,
  though formally covering all biological systems.
  Refers especially to the computational analysis
  of large datasets of DNA, protein and structural
  data

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Simple gene analysis

• Restriction maps
• Plasmid maps
• ORFs and coding sequences
• Database searching
• 2 way comparison of sequences
• Multiple sequence alignments

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More complex analysis

• Sequencing project assembly
• ORF prediction on statistical analysis of DNA
  sequence
• Domain identification
• Structure comparison
• Structure prediction
• Promoter and splice junction prediction
• Genome analysis

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Pairwise alignments

• Comparison of two sequences, DNA-DNA or
  Protein-Protein
• Identification of best matching region
• Local alignment of best match and surrounding
  homology
• Score for similarity and gapping
• Display as alignments or dotplots
• Computational requirement increase as product of
  length of sequences (L1 x L2)

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Algorithms

• Most frequently used methods of alignment are
• Needleman and Wunch
• Smith and Waterman
• FASTA (Pearson)
• BLAST
• Blast and Fasta make approximations to achieve
  speed of alignment/database searching

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Gap insertion costs

• Score for insertion of a gap into a sequence
• Score for extension of a gap into a sequence
• Score for end alignments
• Vary insertion/extension parameters to optimise
  alignments depending on the similarity of the
  sequences
• Lower penalties gives more gaps

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Similarity Scoring Matrices

• Matrices used to score replacement of characters
  in a sequence
• DNA matrix, can be binary, or more complex
• Protein matrix, either binary, or based on
  mutation analysis, biophysical data or other
  easy of replacement options

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Identity DNA scoring matrix

• 1 for identity, 0 for mismatch

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Complex DNA scoring matrix

• For Example
• 4 for identity, 2 for transition, 0 for
  transversion

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Protein Matrices

• Identity matrix 1 for identify, 0 for mismatch
• Mutation data matrix derived from the analysis
  of the easy of substitution of one amino acid for
  another in protein evolution
• Biophysical data matrix based on analysis of
  energy cost of substituting one residue for
  another in protein structures, solubility
  analysis, etc

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Mutation Data Matrix

• PAM - Point Acceptable Mutation various levels,
  differing sensitivities, derived from families of
  ancient, globular proteins
• Blosum - constructed directly from multiple
  sequence alignments. Gives results closer to
  observed relationships. But no evolutionary model.

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PAM 250 Matrix (partial)

• Positive scores allow alignment
• Negative scores indicate poor alignment
• Larger is better (or worse)
• Scale is Logarithmic

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BLOSUM Matrix

• Positive scores allow alignment
• Negative scores indicate poor alignment
• Larger is better (or worse)
• Scale is Logarithmic

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Matrix Comparison

• Selection of matrix depends on extent of
  similarity expected or of interest

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Blast Resources

• Blast Information and resource links
• Blast Tutorial
• Statistics of Similarity Searching
• General Rules for understanding results
• Glossary of technical terms

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BLAST

• Basic Local Alignment and Search Tool
• For rapid searching of pre-processed databases
• 5 search strategies
• Selection of databases and search targets
• Calculated best local match
• Gapped alignments
• Statistics and histograms

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Scoring an alignment
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BLASTN

• Compares Nucleotide sequence with Nucleotide
  Sequence Database
• Complete range of DNA databases
• Use to identify identity or close relationship
  between query sequence and database.
• Not especially sensitive for identification of
  homology
• Good for identity matching

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Nucleotide databases 1

• Nr - non redundant - Database of all published
  and much unpublished DNA sequence data, merged
  from Genbank, EMBL, DDBJ and PDB
• Month - data added to nr within the last month
• Gss - genome sequence survey preliminary genomic
  sequencing project data
• HTGS - high throughput genome sequences,
  unfinished genomic sequences, assembled

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Nucleotide databases 2

• EST - Expressed sequence tags
• Human EST - Human Expressed sequence tags
• Mouse EST - Mouse Expressed sequence tags
• Other EST - Expressed sequence tags from all
  other species
• Other databases E.coli, Yeast, Mito, pbd, kabat,
  patents, vector, alu

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BLASTP

• Protein sequence against protein database
• Complete range of Protein databases
• Use to identify identity and distant relationship
  between query sequence and database.
• Sensitive for identification of homology
• Implementation as PSI-BLAST improves sensitivity
• Good for identity matching

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Protein Databases

• Nr - non redundant - all available data from
  protein and DNA sequence
• Month - most recent 30 days updates
• Swissprot - curated and annotated database
• PDB - protein sequences for 3D structures
• E.coli - E.coli
• Yeast - Saccharomyces
• Kabat - immunological sequences

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TBLASTN

• Compares a protein query sequence against a
  nucleotide sequence database translated into all
  reading frames.
• Sensitive ( as with BLASTP) for homology and
  identity
• Used to identify possible coding sequences and
  homologies
• Especially useful with genome and EST data

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BLASTX

• Compares a nucleotide query sequence translated
  in all reading frames against a protein sequence
  database.
• Sensitive for homology and identity matching
• You could use this option to find potential
  translation products of an unknown nucleotide
  sequence.
• Useful for new DNA sequences

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TBLASTX

• Compares the six-frame translations of a
  nucleotide query sequence against the six-frame
  translations of a nucleotide sequence database.
• Very useful for homology searching
• Less useful with identities (matches everything 6
  times!)
• Helps to select out conserved coding sequence
  from non-coding background
• Especially useful for cross-species analysis with
  genomic, cDNA and EST data

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Blast Inputs

• Input sequence or accession number
• Choice of search program
• Choice of subsequence
• Choice of database
• Choice of codon table

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Blast Input Page
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Blast Matrix choice
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Species/Genus/Phyla selection
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Further species selection
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Boolean operators

• Logical operators used to specify selection
• AND, OR, NOT, (IS, BEFORE, NEXT TO)
• Use to get greater specificity of selection
• For example
• Mammal NOT Human
• Limits selection to all mammals, but excluding
  humans.
• Vertebrate NOT mammal
• Would select all non-mammalian vertebrates

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Format Submission
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Email submission
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Blast Output - summary
Input sequence, name and size
DATABASE - number of sequences, total length
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Blast Output - Graphical
Regions of homology in matching sequences Colour
coded for scores
Input sequence scale
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Blast Output - lists and stats
Database entry
Description of entry
Score
Stats
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Blast Statistics

• Statistical values calculated relative to the
  size of the database
• Depend on the length of the match
• Values expressed as exponentials
• 3e-17 is 3 x 10-17
• Smaller E value is better match, because
  statistically less likely to be a random event
• Exact match has E0 (cannot be random)
• Values greater than 10-5 are questionable

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Blast alignments - exact match
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Blast alignments - homology
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BLAST-2-Sequences

• Pairwise comparison of two sequences only
• All 5 versions of BLAST available
• (so all combinations of DNA/Protein possible)
• Graphical display
• Sequence alignments
• Statistical significance
• calculated from database
• size

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Statistical Significance and Histograms

• Start with Tutorial on
• The Statistics of Sequence Similarity Scores
• Covers matrices, gapping, global vs local
  alignment, statistical significance.

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Multiple Sequence alignments

• Alignment of three more sequences together
• Cannot do alignments simultaneously (excessively
  large computational problem)
• So various options used to develop a rapid
  strategy to align sequences
• Best option to align all sequences as pairs, then
  build multiple sequence alignments added most
  closely related sequences in order

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Clustal approach to MSA

• Compare all sequences with each other
• Pair each sequence with the closest partner
• Align closest partners
• Align next closest partner to create groups
• Align groups of sequences until completed
• Build phylogenetic tree
• Efficient method because only do pairwise
  alignments, and only align closest pairs.

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Outputs from MSA analysis

• Sequence alignment
• Or frequency matrix (Profile)
• Similarity plot
• Phylogenetic tree
• Applies to all programs used for MSA

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Frequency matrix or Profiles

• First matrix created from aligned protein
• Multiplied by mutation data matric (PAM or
  BLOSUM)
• Creates a matrix which is a frequency weighted
  matrix specific to the alignment of sequences
• Provides very sensitive alignment tool
• Can do similarly for DNA sequences

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DWNN domain alignment
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Phylogenetic trees

• Way to show the best predicted evolutionary
  relationship between aligned sequences
• Confidence level depends on method used
• Should relate to evolutionary distance between
  sequences
• Display distances as length and position of
  branches
• Should show up orthologs and paralogs
• Need to root trees correctly for them to give
  correct picture
• Very distance sequences hard to be certain of
  order

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DWNN as a phylogenetic marker
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PSI-BLAST

• Position Specific Iteration BLAST
• Starts with a BLASTP search
• Generates a set of matches
• Select matches above a threshold
• Align sequences scoring above threshold
• Create a frequency matrix from this alignment
• Search database with frequency matrix
• Repeat 1-5 until no new sequences added above
  threshold level

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Advantages of PSI-BLAST

• Generates an alignment from single starting
  sequence
• Creates a specific matrix for each search
  strategy
• Final matrix should be the same for any family of
  proteins, which ever the starting sequence used
  for the search
• Much more sensitive then BLASTP alone

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Current disadvantages of PSI-BLAST

• Does not show alignment used to generate matrix
• Does not show matrix
• Cannot generate final sequence alignment
• May generate several alignments at one time if
  have several domains in the the protein
• Only uses BLASTP, not TBLASTN or BLASTX, so
  databases restricted to protein

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Databases of genomics data

• Databases of genomic sequence data and
  predicted/known genes (eg NCBI Genomes)
• Annotated databases, integrating genetic map,
  physical map (clones), sequence data, known
  genes, and predicted genes
• Databases based on AceDB
• Integration of physical and genetic mapping data
• Common interface for genomics data

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Databases of research papers

• Many different sources
• NCBI PubMed is major site of medical and
  molecular data. But is missing many plant and
  agricultural papers.
• Agricola Agricultural/Biological
• Current Contents Everything!
• www.sciencedirect.com journals from Elselvier
  press, currently free to UWC
• Others at UWC library web site

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Search Engines

• Various types and strategies
• Web based spiders, crawlers etc (Google, Excite,
  Yahoo, Altavista)
• Database based PubMed etc
• PubMed provides comprehensive indexing
• Internally compared to give related references
• Linked access to sequences and literature sites

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Search Strategies

• Need to chose keywords carefully
• Use author names when possible
• Can sometimes select by dates
• Use review as a search term when appropriate
• Add more terms to get greater selectivity
• Avoid general terms, like cell human gene
• Use Boolean operators (and, or, not)
• Look for related articles (in PubMed) based on
  internal text comparison to find most related
  papers

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Genome annotation engines

• Input DNA sequence data
• Search with databases and predictive methods to
  identify possible coding sequences, promoters,
  splice junctions, exons, poly A sites, tRNA
  genes, repeat sequences,
• Some sites do all of this, some dedicated to one
  type of analysis (eg promoters)