Genomics
Gene expression
• Genome maping
• Genome sequencing
• Genome annotations
Structural
genomics
Nucleus
DNA (Genome)
pre-mRNA
Cytoplasm
• DNA arrays and chips
• (semi) qRT-PCR
• Northern blot + hybrid.
• Transkriptional fusions
mRNA
mRNA (Transcriptome)
Proteins (Proteome)
Metabolites
(Metabolome)
• 2D electrophoresis
Mass spectrometry
Protein sequencing
• Translational fusional
• Immunodetection
• Enzyme activities
• Chromatography
• Mass spectrometry
• NMR
Functional
genomics
History of genomes sequencing
• 1977 bacteriophage øX174 (5386bp, 11 genes)
• 1981 mitochondrial genome (16,568bp; 13 prots; 2 rRNAs;
22 tRNAs
• 1986 chloroplast genome (120,000-200,000bp)
• 1992 Saccharomyces chromosome III (315kb; 182 ORFs)
• 1995 Haemophilus influenzae (1.8Mb
• 1996 Saccharomyces whole genome (12.1Mb; over 600 people
100 laboratories)
• 1997 E. coli (4.6Mb; 4200 proteins)
• 1998 Caenorhabditis elegans (97 Mb; 19,000 genů)
• 2000 Arabidopsis thaliana (115Mb, 25-30,000 genů)
• 2001 mouse (1 year!)
• 2001 Homo sapiens (2 projekty)
• 2005 Pan, rice
• 2006 Populus
Technological improvements
DNA sequencing – principle
(Sanger’s method)
Polymeration from primer in the presence of low concentration of terminator
(dideoxy) ddNTP
primer
Random termination on all positions with occurance of the nucleotide
Original arrangement
sequence
- RI labelled primer
- 4 separated reactions
- with individual ddNTP
- ddNTP:dNTP (cca 1:20 – (100))
- PAGE separation
A T C G
C
T
G
G
A
T
C
T
A
G
C
Separation by size
Automated sequencing with
fluorescence-labelled ddNTP
• Every ddNTP labelled with different fluorescent
•
dye – all together in one reaction
Separation by size in capillary – fluorescence
detection
Genom sequencing is more
than sequencing of DNA
• 1 sequencing reaction 300 – 800 bp
• Typical genom hunderts of millions to
billions bp
How to manage?
Strategies of genome sequencing
• Classical strategy (Map-Based Assembly):
- minimal quantity of DNA sequencing
– sorting of big DNA fragments, successive reading
(human genome sequencing – original strategy)
- scaffold for genome sequence assemble
- time consuming
• Whole genome shotgun (WGS)
– random (7-9x redundant) sequencing
– sorting of sequence data (Haemophilus)
- problems with repetitive DNA
• Combination – „hierarchical shotgun“, „chromosome
shotgun“
Hierarchical shotgun sequencing
Whole-genome shotgun sequencing
Production of overlapping clones
(e.g. BACs, YACs)
and construction
of physical map
Shearing of DNA
and sequencing
of subclones
Assembly
Green (2001) Nature Reviews Genetics 2: 573-583
Hierarchical shotgun sequencing
First step: library of big DNA inserts
(= genome fragments)
•
•
•
•
phage (l) vectors: 30 kb
cosmids: 50 kb
BACs (bacterial artificial chromosomes):
100-300 kb
YACs (yeast artificial chromosomes):
cca 0.5-1Mb
Physical „BAC“ map of genome
• Arrangement (position, orientation) of
individual BAC in the genome
• Fundamental for classical sequencing
• Very usefull for assembly of „shotgun“
sequences
How to make the map from BACs with
unknown sequence?
Map construction - BAC fingerprinting
Sequencing of DNA ends
Restriction sites
- 10-20x more bp in BACs than in the genome for map
construction (Arabidopsis – 20 000, rice - 70 000)
BAC fingerprinting
ANIMATION of HIERARCHICAL SHOTGUN: http://www.weedtowonder.org/sequencing.html
Minimum tiling path
= the lowest possible set of BACs covering the whole sequence
physical map arrangement and mapping and clone selection
- by restriction fragment analysis
- using terminal sequences and hybridization
- by hybridization with markers with known position in genetic map
Shotgun sequencing
BAC/chromosome/whole genome
random cleavage +
direct sequencing (NGS)
Cosmids (40 Kbp):
sequencing of
clone ends
(known distance
between)
~500 bp
~500 bp
Genome (chromosome, BAC...) assembly
1.
Looking for overlaps in
primary sequences
2.
Assembly to contigs to get
short consensus sequences
3.
Assebly to supercontigs
using the information of
sequence pairs (ends +
distance)
4.
Complete consensus
sequence
..ACGATTACAATAGGTT..
Repetitive sequences and contig
assembly
repetition
?
?
Repetitions are serious problem in assembly,
if they are conserved and longer than sequencing run
Use of markers for whole genome
assembly
(STS – sequence tagged sites = short sequences with known
position on chromosoms)
Supecontigs with scaffold (BAC-end sequences with known distance)
Filling of gaps: shorter clones are better
X
- optimal – libraries with different insert sizes (2, 10, a 50 kbp)
- sequencing the linker clone = filling the gap
What to do with the genome
sequence? To annotate!
• Searching for genes:
–
–
–
–
Automatic prediction of coding seq.
Prediction of introns/exons
Prediction according to related seq.
Confirmation by cDNA and EST
• Prediction of function
– from experimentally characterized homologues
Fragment of GenBank BAC clone annotation
Graphical interface of BAC annotation
Large genomes
alternative strategies of sequencing:
- isolation of individual chromosomes
e.g. wheat – allows assembly of homeologous
chromosomes (allohexaploid)
- shotgun sequencing of non-methylated DNA (maize)
- sequencing of ESTs (potato)
Expressed Sequence Tags (ESTs)
-short sequenced regions of cDNA (300-600 nt)
-usually gene fragments (primarilly originate from mRNA)
-highly redundant, but also incomplete!
-problems:
- no regulatory sequences (promotors, introns,...)
- only transcripts of certain genes
Expressed Sequence Tags (ESTs)
Preparation of EST library
- mRNA
- RT with oligoT primer  cDNA
-cleavage of RNA from heteroduplex
RNAseH
- 2nd strand cDNA synthesis
- cleavage with restriction endonuclease
- adaptor ligation
cloning
sequencing
Assembly of EST contigs - Unigenes
Next generation sequencing
- faster and cheaper!!!
- parallel sequencing of high numbers of sequences!
- no handling with individual sequences!
Examples of recently developed or developing technologies:
454 sequencing – pyrosequencing (Roche)
- complementary strand synthesis
Illumina – sequencing by synthesis
- complementary strand synthesis
SOLiD - Sequencing by Oligonucleotide Ligation and Detection
- ligation of labelled oligonucleotides
Oxford nanopore technology
- exonuclease degradation, el. current changes detection
NGS – comparison of basic parameters
Method
Single-molecule
real-time
sequencing
(Pacific Bio)
Ion
Sequencing by
semiconductor Pyrosequencing
synthesis
(Ion Torrent
(454)
(Illumina)
sequencing)
Read length
5.000-10.000
(30.000) bp
up to 400 bp
Reads per run
50.000
Cost per 1
million bases (in
US$)
$0.33-$1.00
700 bp
Sequencing by
ligation (SOLiD
sequencing)
Chain
termination
(Sanger
sequencing)
50 to 300 bp
50+50 bp
up to 80 million 1 million
up to 3 billion
1.2 to 1.4 billion N/A
$1
$0.05 to $0.15
$0.13
$10
400 to 900 bp
$2400
http://en.wikipedia.org/wiki/DNA_sequencing
454 technology - pyrosequencing
up to 1 mil reads (lenght 700 - 1000 bp)
one day (23 hour procedure) = 500-800 Mbp
454 technology - pyrosequencing
454 technology
454 technology
Illumina – sequencing by synthesis (Solexa)
Illumina – seqencing by synthesis (Solexa)
Illumina – seqencing by synthesis (Solexa)
Illumina – seqencing by synthesis (Solexa)
SOLiD™ System (Applied Biosystems)
2 Base Encoding Sequencing by Oligonucleotide
Ligation and Detection
- reads up to 75 b
- 20-30 Gb for a day!
- high accuracy up to 99,99 %
- initial step – clonal multiplication (similar to 454)
http://appliedbiosystems.cnpg.com/Video/flatFiles/699/index.aspx
SOLiD™ System
Mix of 1024 octamers (number of variations NNN = 64) x 16 known dinucleotides
Z = nucleotides universally pairing with any nucleotide (prolongation) – cleaved out after ligation
labelling: 4 fluorescent dyes
– each for 256 octamers (with just 4 known middle dinucleotides)
-
5 independent reactions = each 10 – 15 times repeated ligations
of labelled octamers starting from a primer with shifted end
Knowledge of the first nucleotide allows
translation of color sequence to nucleotide sequence
AAT G CA
GGCATG
CCGTAC
}
alternative translation with different 1st nucleotide
Oxford nanopore technologies – direct sequencing
http://www.nanoporetech.com/sequences
of one DNA strand
- protein nanopore in membrane
(alpha-hemolysin)
- covalently bound exonuclease
- monitoring specific decrease
in current (metC!)