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Bioinformática
y Genómica
Central dogma of molecular biology
DNA
genome
RNA
transcriptome
protein
proteome
Central dogma of bioinformatics and genomics
Central dogma of molecular biology
DNA
genome
30,000 genes
RNA
protein
transcriptome
proteome
15,000 transcripts/cell
>>15,000 proteins/cell
Integrative biology in the postgenomic era
DNA
RNA
Protein
Informational pathways
Informational networks
Cells
Organs
Individuals
Populations
Ecologies
Integrative biology in the postgenomic era
DNA
RNA
Protein
Informational pathways
Informational networks
Cells
Organs
Individuals
Populations
Ecologies
Systems biology
Cell biology
Medicine, physiology
Medicine
Genetics
Ecology
Integrative biology in the postgenomic era
DNA
RNA
Protein
Informational pathways
Informational networks
Cells
Organs
Individuals
Populations
Ecologies
Genomics
Functional genomics
Proteomics
Metabolomics
Systems biology
Cell biology
Medicine, physiology
Medicine
Genetics
Ecology
Genómica:
Genómica Funcional
Genómica Estructural
Genómica Funcional
-- Analysis of RNA: gene expression and microarrays
-- Overview of proteins and proteomics
DNA
RNA
cDNA
protein
phenotype
Functional Genomics
Gene expression is regulated
in several basic ways
• by region (e.g. brain versus kidney)
• in development (e.g. fetal versus adult tissue)
• in dynamic response to environmental signals
(e.g. immediate-early response genes)
Genomics Technologies
• Automated DNA sequencing
• Automated annotation of sequences
• DNA microarrays
– gene expression (measure RNA levels)
– single nucleotide polymorphisms (SNPs)
The human genome is the the complete DNA
content of the 23 pairs of human chromosomes
- 44 autosomes plus two sex chromosomes
- approximately 3.5 billion base pairs.
How does genome
sequencing technology
work?
• Molecular biology of the Sanger method
• Manual Gels vs. ABI machines
• Sub-cloning of fragments - BAC, PAC,
cosmid, plasmid, phage
• The need for computers to assemble the
"reads" and manage the workflow
Automated sequencing machines,
particularly those made by PE Applied
Biosystems, use 4 colors, so they can
read all 4 bases at once.
All the Genes?
• Any human gene can now be found in the
genome by similarity searching with over
95% certainty.
• However, the sequence still has many
gaps
– unlikely to find an uninterrupted genomic
segment for any gene
– still can’t identify pseudogenes with certainty
• This will improve as more sequence data
accumulates
Finding Genes in genome
Sequence is Not Easy
• About 2% of human DNA encodes
functional genes.
• Genes are interspersed among long
stretches of non-coding DNA.
• Repeats, pseudo-genes, and introns
confound matters
Impact on Bioinformatics
• Genomics produces high-throughput, highquality data, and bioinformatics provides
the analysis and interpretation of these
massive data sets.
• It is impossible to separate genomics
laboratory technologies from the
computational tools required for data
analysis.
Billions of years ago (BYA)
Origin of Earliest
life
fossils
Hadean
eon
Origin of
eukaryotes
Archean eon
4
3
insects
Fungi/animal
Plant/animal
Phanerozoic
eon
Proterozoic eon
2
1
0
Millions of years ago (MYA)
deuterostome/
echinoderm/
protostome Insects
chordate
Cambrian Land
explosion plants
Proterozoic eon
1000
Age of
Reptiles ends
Phanerozoic eon
500
100
0
Millions of years ago (MYA)
Mass
extinction
100
Human/chimp
divergence
Dinosaurs extinct;
Mammalian radiation
50
10
0
Millions of years ago (MYA)
Homo Sapiens/
Chimp divergence
10
Australepithecus Earliest
Lucy stone tools
5
Emergence of
Homo erectus
1
0
Years ago
Homo erectus
emerges in Africa
1,000,000
Mitochondrial
Eve
500,000
100,000
Years ago
Emergence of
anatomically
modern H. sapiens
100,000
Neanderthal and Homo
erectus disappear
50,000
10,000
Years ago
“Ice Man” Earliest
from Alps pyramids
10,000
5,000
1,000 0
prominent
archaea
bacteria
eukaryota
http://www.ncbi.nlm.nih.gov/Entrez/
Overview of viral complete genomes
Overview of archaea complete genomes
Overview of eukaryota genomes in NCBI’s Entez division
Chronology
1977
Sanger et al. sequence bacteriophage fX174
1981
Human mitochondrial genome
16,500 base pairs
1986
Chloroplast genome
156,000 base pairs
mitochondrion
chloroplast
Lack
mitochondria (?)
1995: genome of the bacterium
Haemophilus influenzae is sequenced
Overview of bacterial
complete genomes
COGs database:
organisms and tools
COGs database:
functional annotation
1996: a yeast genome is sequenced
Key yeast databases
Saccharomyces genome database (SGD)
http://genome-www.stanford.edu/Saccharomyces/
MIPS Comprehensive Yeast Genome Database
(MIPS = Munich Information Center for Protein Sequences)
http://mips.gsf.de/proj/yeast/CYGD/db/
PomBase -A S. pombe ACeDB Database
http://www.sanger.ac.uk/Projects/S_pombe/pombase.shtml
Genome sizes in nucleotide base pairs
plasmids
viruses
bacteria
fungi
plants
algae
insects
mollusks
bony fish
The size of the human
genome is ~ 3 X 109 bp;
almost all of its complexity
is in single-copy DNA.
amphibians
reptiles
birds
The human genome is thought
to contain ~30,000-40,000 genes.
104
105
106
107
mammals
108
109
1010
1011
http://www3.kumc.edu/jcalvet/PowerPoint/bioc801b.ppt
eukaryotes
1999: Human chromosome 22 sequenced
1999: Human chromosome 22 sequenced
47 MB
780 genes
2000
Completed genome projects
Eukaryotes: 9
In progress (partial):
Anopheles gambiae
Danio rerio (zebrafish)
Arabidopsis thaliana
Glycine max (soybean)
Caenorhabditis elegans
Hordeum vulgare (barley)
Drosophila melanogaster
Leishmania major
Encephalitozoon cuniculi
Rattus norvegicus
Guillardia theta nucleomorph
Plasmodium falciparum
Saccharomyces cerevisiae (yeast)
Schizosaccharomyces pombe
Bacteria: 132
Archaea: 16
Viruses: 1413
eukaryotes
Six basic questions about genomes
[1] how is a genome sequenced?
[2] when is the project finished?
[3] sequence one individual or many?
[4] what information is in the DNA?
[5] how many genes are in the genome?
[6] how can whole genomes be compared?
[1] Genome projects: sequencing strategies
Hierarchical shotgun method
Assemble contigs from various chromosomes, then sequence and assemble them. A contig
is a set of overlapping clones or sequences from which a sequence can be obtained. The
sequence may be draft or finished.
A contig is thus a chromosome map showing the locations of those regions of a
chromosome where contiguous DNA segments overlap. Contig maps are important
because they provide the ability to study a complete, and often large segment of the genome
by examining a series of overlapping clones which then provide an unbroken succession of
information about that region.
Scaffold: an ordered set of contigs placed on a chromosome.
Shotgun
An approach used to decode an organism's genome by shredding it into smaller
fragments of DNA which can be sequenced individually. The sequences of these
fragments are then ordered, based on overlaps in the genetic code, and finally
reassembled into the complete sequence. The 'whole genome shotgun' method is
applied to the entire genome all at once, while the 'hierarchical shotgun' method is
applied to large, overlapping DNA fragments of known location in the genome.
http://www.genome.gov/glossary.cfm
3. Whole Genome Shotgun
Sequencing
genome
cut many times at
random
• plasmids (2 – 10 Kbp)
• cosmids (40 Kbp)
~500 bp
forward-reverse
linked reads
known dist
~500 bp
ARACHNE:
Whole Genome Shotgun Assembly
1. Find overlapping reads
2. Merge good pairs of reads
into longer contigs
3. Link contigs to form
supercontigs
4. Derive consensus sequence
..ACGATTACAATAGGTT..
http://www-genome.wi.mit.edu/wga/
[2] When is the project finished?
Get five to ten-fold coverage
Finished sequence: a clone insert is contiguously
sequenced with high quality standard of error rate
0.01%. There are usually no gaps in the sequence.
Draft sequence: clone sequences may contain several
regions separated by gaps. The true order and
orientation of the pieces may not be known.
[3] Sequence one individual or many?
Try one…
--Each genome center may study 1 chromosome
--Measure polymorphisms (e.g. SNPs) in
large populations
[4] What information is in the DNA?
-- repetitive DNA elements
-- nucleotide composition (GC content)
-- protein-coding genes, other genes
Genome sizes in nucleotide base pairs
plasmids
viruses
bacteria
fungi
plants
algae
insects
mollusks
bony fish
The size of the human
genome is ~ 3 X 109 bp;
almost all of its complexity
is in single-copy DNA.
amphibians
reptiles
birds
The human genome is thought
to contain ~30,000-40,000 genes.
104
105
106
107
mammals
108
109
1010
1011
http://www3.kumc.edu/jcalvet/PowerPoint/bioc801b.ppt
DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
k2
Slower, rate-limiting,
second-order process of
finding complementary
sequences to nucleate
base-pairing
Faster,
zippering
reaction to
form long
molecules
of doublestranded
DNA
http://www3.kumc.edu/jcalvet/PowerPoint/bioc801b.ppt
% DNA reassociated
Britten and Kohne (1968) provided evidence for
large amounts of repetitive DNA in many genomes
0
fast ~10%
intermediate
~15%
50
slow (single-copy)
~75%
100
I
I
I
I
I
log Cot
I
I
I
I
Repetitive DNA sequences: five classes
[1] Interspersed repeats: transposon-derived repeats
-- 45% of human genome; LTR, SINE, LINE
[2] Processed pseudogenes
[3] Simple sequence repeats
-- micro- and minisatellites
-- ACAAACT, 11 million times in a Drosophila
-- Human genome has 50,000 CA dinucleotide repeats
[4] Segmental duplications (about 5% of human genome)
[5] Tandem repeats (e.g. telomeres, centromeres)
• LINE and SINE repeats. A LINE (long interspersed
nuclear element) encodes a reverse transcriptase (RT) and
perhaps other proteins. Mammalian genomes contain an
old LINE family, called LINE2, which apparently stopped
transposing before the mammalian radiation, and a
younger family, called L1 or LINE1, many of which were
inserted after the mammalian radiation (and are still being
inserted). A SINE (short interspersed nuclear element)
generally moves using RT from a LINE. Examples include
the MIR elements, which co-evolved with the LINE2
elements. Since the mammalian radiation, each lineage has
evolved its own SINE family. Primates have Alu elements
and mice have B1, B2, etc. The process of insertion of a
LINE or SINE into the genome causes a short sequence (721 bp for Alus) to be repeated, with one copy (in the same
orientation) at each end of the inserted sequence. Alus have
accumulated preferentially in GC-rich regions, L1s in GCpoor regions.
What is the function of nongenic DNA?
Hypotheses:
• Nongenic DNA performs essential functions, such as
regulation of gene expression.
• Nongenic DNA is inert, genetically and physiologically.
Excess DNA is incidental and is called “junk DNA.”
• Nongenic DNA is a functional parasite or selfish DNA
(retrotransposons).
• Nongenic DNA has a structural function.
GC content varies across genomes
Bacteria
Number of species
in each GC class
10
5
Plants
5
Invertebrates
3
Vertebrates
10
5
20
30
40
50
60
70
GC content (%)
80
[5] How many genes are in the a genome?
This depends how a gene is defined (e.g. proteincoding versus noncoding)
It also depends what methods are used to find genes,
and what criteria are applied to determine whether
they are “real” (functional).
Clasificación del ADN
FUNCIONAL (secuencias que cumplen una función)
- Codante (se traducen en proteínas)
-No codante (no se traducen)
* Transcrito (cumple función a nivel de RNA: subun. ribos.)
* No transcrito (cumple función a nivel de DNA: intrón,
promotor, enhancer, etc.)
NO-FUNCIONAL (secuencias que no cumplen ninguna función: “Junk DNA” –
basura)
Gene-finding algorithms
Homology-based searches (“extrinsic”)
Rely on previously identified genes
Algorithm-based searches (“intrinsic”)
Investigate nucleotide composition, openreading frames, and other intrinsic
properties of genomic DNA
DNA
intron
RNA
Mature RNA
protein
Homology-based searching: compare DNA
to expressed genes (ESTs)
DNA
intron
RNA
RNA
protein
DNA
RNA
Algorithm-based searching: compare DNA in exons
(unique codon usage) to introns (unique splices sites)
to noncoding DNA. Identify open reading frames (ORFs).
[5’] How many genes are in the human genome?
One answer is about 30,000. BUT how many genes?…
-- A lot more than a fungus (6,000)
-- Somewhat more than a fly (13,000) or a worm
(19,000)
-- About the same as a plant (Arabidopsis, 25,000)
-- Two groups estimate 30,000 to 35,000, but there is
only partial overlap in their gene lists!
-- One Drosophila gene potentially yields 38,000
distinct proteins by alternative splicing.
-- A microarray-based survey of chromosomes 21, 22
finds 10 times more transcripts than are annotated
[6] how can whole genomes be compared?
-- molecular phylogeny
-- You can BLAST (or PSI-BLAST) all the DNA and/or
protein in one genome against another
-- We looked at TaxPlot and COG for bacterial (and for
some eukaryotic) genomes
-- PipMaker and other programs align large stretches of
genomic DNA from multiple species
Resources to study the human genome
NCBI
www.ncbi.nlm.nih.gov
The Sanger Institute/European Bioinformatics Institute
www.ensembl.org
UCSC Genome Bioinformatics Site
http://genome.ucsc.edu/
Top ten challenges for bioinformatics
[1] Precise models of where and when transcription
will occur in a genome (initiation and termination)
[2] Precise models RNA splicing
[3] Precise models of signal transduction pathways;
ability to predict cellular responses to external stimuli
[4] Determining protein:DNA, protein:RNA, protein:protein
recognition codes
[5] Accurate ab initio protein structure prediction
Top ten challenges for bioinformatics
[6] Rational design of small molecule inhibitors of proteins
[7] Mechanistic understanding of protein evolution
[8] Mechanistic understanding of speciation
[9] Development of effective gene ontologies:
systematic ways to describe gene and protein function
[10] Education: development of bioinformatics curricula
Comparative Genomics
Using ACT
The Artemis Comparison Tool
Artemis comparison tool ACT
• Based on artemis and coded in java.
• Allows visualisation of two sequences or
more and a comparison file.
• The comparison file can be BLASTn or
tBLASTx.
• Retains all the functionality of artemis.
The ACT Display
genome1
Zoom scroll bar
Filter scroll
bar
genome2
Genome2
Blast HSPs
genome3
Running ACT
Sequence 1
Sequence 2
BLASTn
tBLASTx
MSPcrunch
Reformat
ACT
• Designed for looking at complete bacterial
genomes.
Knowlesi
contgs
tblastx
Falciparum
Chr 3
tblastx
Yoelii
Contigs (TIGR)
Orthologue & Paralogue
• Orthologue- homologous genes with
identical function in different organisms.
• Paralogue- homologous genes in the same
organism originated from gene duplication.
Orthologue & Paralogue
Species 1
Species 2
Gene A
Gene A
Gene B
Gene B
diverge
Orthologue & Paralogue
Species 1
Species 2
Gene A
Gene A
Gene B
Gene B
Orthologue & Paralogue
Species 1
Species 2
Gene A
Gene A
Gene B
Orthologue & Paralogue
Species 1
Species 2
Gene A
Gene B
T. brucei vs L. major (cont.)
T. brucei vs T. cruzi
L. major has break in synteny that is
conserved in T. brucei and T. cruzi
T. cruzi
Chr3.
T. Brucei
chr1
T. Brucei
chr6
L. Major
chr12
Software
• www.sanger.ac.uk/Software/Artemis
• www.sanger.ac.uk/Software/ACT
• www.genome.nghri.nih.gov/blastall
• www.cgr.ki.se/cgr/goups/sonnhammer/MSPcrunch.html