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
