How to use the web for bioinformatics
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How to use the web for bioinformatics. Ethan Strauss ethan.strauss@promega.com 274-4330 X 1171 http://www.q7.com/~ethan. 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:

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How to use the web for bioinformatics

How to use the web for bioinformatics

Ethan Strauss

ethan.strauss@promega.com

274-4330 X 1171

http://www.q7.com/~ethan


Objectives

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


How to find all those dang urls
How to find all those dang URLs!

  • http://q7.com/~ethan/molbio/


Outline
Outline

  • Sequence Databases

    • What does a Genbank Entry look like?

  • Translation and other Utilities

  • BLAST

  • Multiple Sequence Alignment

  • PCR Primer Design


Sequences databases
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.


Sequences databases ncbi genebank
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!


Sequences databases entrez
Sequences DatabasesEntrez

  • Allows searching and access to NCBI databases.


Sequences databases sequence records
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=nucleotide&val=58533118


Hands on
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


General utilities
General Utilities

  • http://searchlauncher.bcm.tmc.edu/seq-util/seq-util.html

    • Translation

    • Restriction Digestion

    • Reformatting (alternately FASTA Formatter)

    • Complement/Reverse

    • Etc.

  • http://www.promega.com/biomath/calc11.htm

    • Melting Temperature of an oligo.


Hands on1
Hands on

  • Translate your sequence in all 6 reading frames.


Blast
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




Hands on2
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.


Multiple sequence alignment
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.


Multiple sequence alignment1
Multiple Sequence Alignment

  • MEAGAYLNAIIFVLVATIIAVISRGLTRTEPCTIRITGESITVHACHIDSX ETIKALA MEAGAYLNAIIFVLVATIIAVISRGLTRTEPCTIRITGESITVHACHIDS...ETIKALA MEA..YLNAII.VLV.TIIAVIS..L.RTEPC.IkITGESITV.ACklDa.....I..L. MEAgaYLNAIIfVLVaTIIAVISrgLtRTEPCtIrITGESITVhAChiDsx etIkaLa

  • LK PLSLERLFQ LK.PLSLERLFQ ......L..... lk plsLerlfq


Hands on3
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


Pcr primer design
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.


Hands on4
Hands on

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


Homework
Homework

  • Report:Please 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.