Bioinformatics:

Copyright© Kerstin Wagner

 Introduction: What is bioinformatics?
Can be defined as the body of tools, algorithms needed to handle large
and complex biological information.

Bioinformatics is a scientific discipline created from the interaction

of biology and computer science.

The NCBI defines bioinformatics as:

"Bioinformatics is the field of science in which biology, computer
science, and information technology merge into a single discipline”
 Genomics era: High-throughput DNA sequencing

The first high-throughput genomics

technology was automated DNA sequencing
in the early 1990.

In 1995, Venter and Hamilton used whole-

genome shotgun sequencing strategy to
sequence the genomes of Mycoplasma and
Haemophilus .

In September 1999, Celera Genomics

completed the sequencing of the
Drosophila genome.

The 3-billion-bp human genome sequence

was generated in a competition between
the publicly funded Human Genome
Project and Celera
 High-throughput DNA sequencing

Top image: confocal detection

by the MegaBACE sequencer
of fluorescently labeled DNA

That was then. How about

now?
 The trend of data growth
21st century is a century of biotechnology and OMICS:
8
7

Nucleotides(billion)
6
5
 Genomics: New sequence information is being 4
3
produced at increasing rates. (The 2
contents of GenBank double every year) 1
0
1980 1985 1990 1995 2000

 Transcriptomics: Microarray: Global expression analysis: RNA Years

levels of every gene in the genome analyzed in parallel.

Progressively replaced by RNA-seq

 Proteomics: Global protein analysis generates by large mass

spectra libraries.

 Metabolomics: Global metabolite analysis: 25,000 secondary

metabolites characterized
How to handle the large amount of information?

Drew Sheneman, New Jersey--The Newark Star Ledger

Answer: bioinformatics and Internet

 Bioinformatics history
In1960s: the birth of bioinformatics

IBM 7090 computer

Margaret Oakley Dayhoff created:

The first protein database
The first program for sequence assembly

There is a need for computers and algorithms that allow:

Access, processing, storing, sharing, retrieving, visualizing, annotating…
 Why do we need the Internet?
“omics” projects and the information associated with involve a huge amount
of data that is stored on computers all over the world.
Because it is impossible to maintain up-to-date copies of all relevant
databases within the lab. Access to the data is via the internet.
 Database
storage

You are
here
 Scope of this lab
The lab will touch on the following computational tasks:
Similaritysearch
Sequence comparison: Alignment, multiple alignment, retrieval
Sequences analysis: Signal peptide, transmembrane domain,…
Protein folding: secondary structure from sequence
Sequence evolution: phylogenetic trees

Make you familiar with bioinformatics resources available on the

web to do these tasks.
 Applying algorithms to analyze genomics data
-Accession #?
-Annotation?
Is it already in
databases?
Protein Other
characteristics? information?
-Sub-localization -Expression profile?
-Soluble? -Mutants?
You have just
-3D fold
cloned a gene

Is there conserved Is there similar Evolutionary

regions? sequences? relationship?
-Alignments? -% identity? -Phylogenetic
-Domains? -Family member? tree

A critical failure of current bioinformatics is the lack of a single software

package that can perform all of these functions.
 DNA (nucleotide sequences) databases
They are big databases and searching either one should produce
similar results because they exchange information routinely.

-GenBank (NCBI): http://www.ncbi.nlm.nih.gov

-Ensembl: http://useast.ensembl.org/index.html

-DDBJ (DNA DataBase of Japan): http://www.ddbj.nig.ac.jp

-TIGR: http://tigr.org/tdb/tgi

-Yeast: http://yeastgenome.org

-Microbes: http://img.jgi.doe.gov/cgi-bin/pub/main.cgi
 Protein (amino acid) databases
Known proteins:
-Swiss-Prot (very high level of annotation)
http://au.expasy.org/

-PIR (protein identification resource) the world's most

comprehensive catalog of information on proteins
http://www.pir.uniprot.org/

Translated databases:
-TREMBL (translated EMBL): includes entries that have
not been annotated yet into Swiss-Prot.
http://www.ebi.ac.uk/trembl/access.html

-GenPept (translation of coding regions in GenBank)

-pdb (sequences derived from the 3D structure

Brookhaven PDB) http://www.rcsb.org/pdb/
 Database homology searching
Use algorithms to efficiently provide mathematical basis of searches
that can be translated to statistical significance.

Assumes that sequence, structure, and function are inter-related.

All
similarity searching methods rely on the concepts of alignment
and distance between sequences.

A similarity
score is calculated from a distance: the number of DNA
bases or amino acids that are different between two sequences.
 Database search methods: Sequence Alignment
Two broad classes of sequence alignments exist:

QKESGPSSSYC
 Global alignment: not sensitive
VQQESGLVRTTC

ESG
 Local alignment: faster
ESG

The most widely used local similarity algorithms are:

Smith-Waterman (http://www.ebi.ac.uk/MPsrch/)
Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nih.gov)

Fast Alignment (FASTA, http://fasta.genome.jp; http://www.ebi.ac.uk/fasta33/;

http://www.arabidopsis.org/cgi-bin/fasta/nph-TAIRfasta.pl)
 Which algorithm to use for database similarity search?

Speed:
BLAST > FASTA > Smith-Waterman (It is VERY SLOW and uses a
LOT OF COMPUTER POWER)

Sensitivity/statistics:
FASTA is more sensitive, misses less homologues
Smith-Waterman is even more sensitive.

BLAST calculates probabilities

FASTA more accurate for DNA-DNA search then BLAST

 Tools to search databases
The dilemma: DNA or protein?

Search by similarity

Using nucleotide seq. Using amino acid seq.

 Is the comparison of two nucleotide sequences accurate?

 By translating into amino acid sequence, are we losing information?

The genetic code is degenerate (Two or more codons can represent
the same amino acid)

 Very different DNA sequences may code for similar protein sequences
We certainly do not want to miss those cases!
 Reasons for translating
Comparing DNA sequences give more random matches:
A good alignment with end-gaps A very poor alignment

Almost 50% identity!

Conservation of protein in evolution (DNA similarity decays faster!)

Conclusion:
It is almost always better to compare coding sequences in their amino acid form,
especially if they are very divergent.
 Very highly similar nucleotide sequences may give better results.
 BLAST and FASTA variants

FASTA: Compares a DNA query to DNA database, or a protein query

to protein database
FASTX: Compares a translated DNA query to a protein database
TFASTA: Compares a protein query to a translated DNA database

BLASTN: Compares a DNA query to DNA database.

BLASTP: Compares a protein query to protein database.

BLASTX: Compares the 6-frame translations of DNA query to protein

database.
TBLASTN: Compares a protein query to the 6-frame translations of a DNA
database. You can however define your frame of interest
TBLASTX: Compares the 6-frame translations of DNA query to the 6-frame
translations of a DNA database (each sequence is comparable to
BLASTP searches!)

PSI-BLAST: Performs iterative database searches. The results from each round
are incorporated into a 'position specific' score matrix, which is
used for further searching
 A practical example of sequence alignment
http://www.ncbi.nlm.nih.gov

BLAST results
 Detailed BLAST results

E value: is the expectation value or probability to find by chance hits similar to

your sequence. The lower the E, the more significant the score.
 Database searching tips
Use latest database version.

Use BLAST first, then a finer tool (FASTA,…)

Search both strands when using FASTA.

Translate sequences where relevant

Search 6-frame translation of DNA database

E < 0.05 is statistically significant, usually biologically

interesting.

If the query has repeated segments, delete them and

repeat search