How to use the web for bioinformatics

Ethan Straussethan.strauss@promega.com

274-4330 X 1171

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

ObjectivesAt 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!

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

Outline

• Sequence Databases– What does a Genbank Entry look like?

• Translation and other Utilities

• BLAST

• Multiple Sequence Alignment

• PCR Primer Design

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 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 DatabasesEntrez

• Allows searching and access to NCBI databases.

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

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

• 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 on

Translate your sequence in all 6 reading frames.

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

BLAST

BLAST

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

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

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

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

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 on

Design primers for the sequence you have been working with.

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.