How to use the web for bioinformatics

1
How to use the web for bioinformatics

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

2
Objectives

• At the end of this session you should be able to
  do all of the following 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 a BLAST server (e.g. ) to find related
  sequences.
• Perform an alignment of several nucleic acid
  sequences
• Obtain the protein sequence which corresponds to
  a specific Nucleic acid sequence

3
How to find all those dang URLs!

• http//q7.com/ethan/molbio/

4
Outline

• Sequence Databases
• What does a Genbank Entry look like?
• Translation and other Utilities
• BLAST
• Multiple Sequence Alignment
• PCR Primer Design

5
Sequences 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.

6
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 a protein database needs!

7
Sequences DatabasesEntrez

• Allows searching and access to NCBI databases.

8
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

9
Hands on

• Find a gene of interest using the Entrez
  interface.
• We will be working with this sequence throughout
  class, so you may want to open a word processing
  program and save the sequence (only) there for
  future reference

10
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.

11
Hands on

• Translate your sequence in all 6 reading frames.

12
BLAST

• Basic Local Alignment Search Tool
• Compares a query sequences against all sequences
  in a database.
• Very powerful for finding biologically
  significant relationships and full gene sequences
  in the database when you have a fragment etc.
• Different types
• Nucleic acid Nucleic Acid
• Protein- Protein
• Nucleic Acid Translation Protein
• Protein Nucleic Acid Translation
• Translation - Translation

13
BLAST
14
BLAST
15
Hands on

• Use 120 bases (2 lines) from your sequence to
  find at least two other sequences related to it.
• Note that if we all hit NCBI BLAST at once, it
  will be slow. We may not have time to wait.
• Get all 3 sequences (your original and two
  others) into FASTA format using READSEQ.

16
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.

17
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

18
Hands on

• Use your FASAT Formatted sequences to perform a
  multiple sequence alignment.
• Transfer the alignment to a word processing
  program and see if you can make it look decent.
• Change to Courier or Courier New
• Reduce Font Size
• Change to Landscape view

19
PCR Primer Design

• There are many PCR primer design programs online
  and off.
• I recommend Primer 3. It is complex, but
  powerful.
• You can ignore most parameters.

20
Hands on

• Design primers for the sequence you have been
  working with.

21
Homework

• ReportPlease turn in a report which includes
  the following
• Information about your initial sequence
  including
• Genebank Accession Number
• Species
• Description
• Location of ORF and any other important features.
  
• Information about the 4 other sequences including
  the above
• Genebank Accession Number
• Species
• Description
• Location of ORF and any other important features.
  
• E value from your BLAST results.
• The sequences of the PCR primers you chose or a
  short explanation of why you could not find
  primers to amplify all of these genes.
• The multiple sequence alignment with the
  locations of the primers clearly marked.