How to use the web for bioinformatics

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How to use the web for bioinformatics

• Molecular Technologies
• Ethan Strauss
• ethan.strauss_at_promega.com
• 274-4330 X 1171
• http//www.q7.com/ethan

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Objectives

• At the end of this session you should be able to
  do all of the following using freely available
  tools on the world wide web
• Use Genbank or a similar database to find nucleic
  acid sequences of interest
• Understand the parts of a Genbank entry
• Use some of the databases at NCBI to find more
  information about a sequence.
• Perform an alignment of several nucleic acid
  sequences
• Find an arbitrary tool or database on the web.

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How to find all those dang URLs!

• http//q7.com/ethan/molbio/

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Outline

• What is Bioinformatics
• Sequence Databases
• What does a Genbank Entry look like?
• Other NCBI databases
• Multiple Sequence Alignment
• New tools Databases

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What is Bioinformatics?

• Bioinformatics refers to the creation and
  advancement of algorithms, computational and
  statistical techniques, and theory to solve
  formal and practical problems posed by or
  inspired from the management and analysis of
  biological data (Wikipedia)

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What is Bioinformatics?(my working definition)

• Anything done on a computer in which knowledge of
  biology is helpful.
• or
• Anything done in biology in which knowledge of
  computers is helpful.

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What sort of questions can Bioinformatics answer?

• Sequence analysis
• Where are restriction sites?
• How does an RNA molecule fold?
• What changes can be made to a DNA sequence to
  get a new protein with specific functional
  changes?
• Computational evolutionary biology
• How are two sequences related?
• Analysis of gene expression
• Is this gene highly expressed in cancer cells?

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What sort of work is done in Bioinformatics?

• Measuring biodiversity
• How diverse are individuals of a species?
• Is it one species or two?
• Analysis of regulation
• What does this drug do to expression of a gene?
• Analysis of mutations in cancer
• What is different about these cancer cells as
  compared to none cancer cells?
• High-throughput image analysis
• How can we analyze the affects of 1000 different
  compounds on the location of a specific protein?
• And more!

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Sequence Databases

• NCBI databases Nucleic acids, proteins,
  Literature, genomes, taxonomy, SNPs and more!
• EMBL Nucleic acid, protein, structure,
  microarray data and more.
• DBJJ Nucleic acid, protein.
• SwissProt Very well annotated protein database.
  
• Many other general and specialized databases
  exist.

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Sequences DatabasesNCBI/Genebank

• Nation Center for Biotechnology Information
  (NCBI)
• Sponsored and run by the US government.
• Contains many different databases and huge
  amounts of information.
• Most or all data is freely downloadable.
• This one site is probably sufficient for all your
  Nucleic acid and Protein database needs!

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Sequences DatabasesEntrez

• Allows searching and access to NCBI databases.

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Sequences DatabasesSequence Records

• LOCUS Number Size Type Topology
  Division Date
• DEFINITION - Name of the Sequence
• ACCESSION - Unique Id number
• VERSION - Other numbers which are associated
• KEYWORDS
• SOURCE What was it isolated from
• ORGANISM - More taxonomic detail
• REFERENCE - Paper or papers about the sequence
• AUTHORS
• TITLE
• JOURNAL
• FEATURES - A complete list of all of the
  features of a sequence. Can be very extensive and
  useful!
• ORIGIN The actual Sequence!
• http//www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db
  nucleotideval58533118

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Other NCBI databases

• Online Mendelian Inheritance in Man (OMIM)
• A catalog of human genes and genetic disorders
  with links to other NCBI databases, including
  sequence databases.
• This is a good starting point if you want to get
  sequences for a specific disorder.
• http//www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD
  searchDBomimtermHFI

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Other NCBI databases

• Gene Database
• Gathers information about a single gene.
• Exactly one entry per Gene.
• A good place to dig deeper into a single gene or
  to reduce redundancy about a single gene.

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Other NCBI databases

• HomoloGeneGathers homologs from various species
• 3D DomainsProtein Structure collection
• TaxonomySpecies information
• Geo (Gene Expression Omnibus)A gene
  expression/molecular abundance repository

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General Utilities

• http//searchlauncher.bcm.tmc.edu/seq-util/seq-uti
  l.html
• Translation
• Restriction Digestion
• Reformatting (alternately FASTA Formatter)
• Complement/Reverse
• Etc.
• http//www.promega.com/biomath/calc11.htm
• Melting Temperature of an oligo.

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Database search by sequence similarity

• Basic Local Alignment Search Tool (BLAST)

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Multiple Sequence Alignment

• Many programs can align multiple sequences with
  each other to find the best fit for all.
• This is generally more biologically meaningful
  for protein sequences since they are more highly
  conserved.
• Clustal is the most common.

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Multiple Sequence Alignment

• MEAGAYLNAIIFVLVATIIAVISRGLTRTEPCTIRITGESITVHACHID
  SX ETIKALA MEAGAYLNAIIFVLVATIIAVISRGLTRTEPCTIRITG
  ESITVHACHIDS...ETIKALA MEA..YLNAII.VLV.TIIAVIS..L.
  RTEPC.IkITGESITV.ACklDa.....I..L.
  MEAgaYLNAIIfVLVaTIIAVISrgLtRTEPCtIrITGESITVhAChiDs
  x etIkaLa
• LK PLSLERLFQ LK.PLSLERLFQ ......L..... lk
  plsLerlfq

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New Tools

• Development of new tools and databases is
  ongoing.
• Your needs will probably change over time.
• You can find new tools using
• Google
• Lists
• Nucleic Acids Research Annual Database issue

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Homework

• Assignments due next session
• Find a entry of interest in OMIM
  (http//www.ncbi.nlm.nih.gov/entrez/query.fcgi?db
  OMIM)
• Find a Gene associated with that entry
• Click on the links link on the right and
  choose Gene

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Homework

1. The Gene page has gathered scads of information
   about this one gene. Find homologs in other
   species. From this page again choose links and
   go to Homologene

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Homework

1. Gather the protein sequences for each homologous
   gene (or 5 of them if there are more than that).
2. Click DownLoad in the homologene listing
   
3. Download everything with the default settings.

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Homework

• You will get a text file in Fasta format. Save
  it somewhere convenient.

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Homework

• Go to the Clustal server at http//searchlauncher.
  bcm.tmc.edu/multi-align/multi-align.html
• Paste your complete Fasta file contents into the
  input box and click submit.
• This takes awhile, so be patient. You will get
  output that looks something like this.

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Homework

• At the bottom of the alignment file is the same
  results in Fasta format. Copy the complete
  Fasta results and paste it into the input box at
  a BoxShade server (http//bioweb.pasteur.fr/seqana
  l/interfaces/boxshade.html)

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Homework

• Depending on the parameters chosen for BoxShade,
  you will see something like this. Regions which
  are the same in all species are likely involved
  in function in some way.

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Homework

• After all that work, your boss comes to you ands
  says that sequence comparison is obsolete! He
  wants you do structural alignments of these
  proteins. Figure out what a structural alignment
  is, find two different tools to find conserved 3D
  structures and choose which one you would use for
  this. Describe why this tool is preferable to the
  other.
• NOTE You do not need to actually do any
  structural alignments. Just find out how you
  would go about doing on if you had to.