Post on 21-Dec-2015
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Large!
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High throughput technologies:
• Sequencing• Gene expression profiling• Chip-CHIP and tiling arrays• Whole genome yeast two hybrid scan• Genomic knockout of all single genes• SNP/CGH• Methylation profiling … • Proteome profiling
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Genomic Sequencing – shotgun sequencing
Sequencing is usually ~700 bp in a single run.
How can we sequence a genome?
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Genomic Sequencing – Walking.
1.Design a primer2.Sequence.3.Design a new primer4.Sequence5.…
One has to design new primers every time. To do so, one has to wait for the sequencing results
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GAGGAGACGAACACCCGTATACAGTCGACG
ACCCCGAGGAGACGAACACCCGTATACAGTCGACGTTTATATATA
GTATACAGTCGACGTTTATATATA
ACCCCGAGGAGACGA
Genomic Sequencing – shotgun sequencing
1. Break DNA to small pieces2. Sequence each piece3. Assemble
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After the DNA is isolated (from the tissue/cell/virus), it is fragmented either by restriction enzymes or by mechanical force.
ACGTAACGTATACCCGACTATATGCATTGCATATG “Frayed ends”
1 .Break DNA to small pieces
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←ATACGTAACGTATACCCGAC
TATATGCATTGCATATGGG→3’
5’
5’
3’
To blunt-end (“fix”) frayed ends, one needs a DNA polymerase. In the example above, just adding a polymerase will make the edges blunt.
Polymerases always make the chain grow from the 5’ towards the 3’ (5’ → 3’)
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ACGTAACGTATACCCGAC ATTGCATATGGGCTGAACAT
3’
5’
5’
3’
Polymerases always make the chain grow from the 5’ towards the 3’ (5’ → 3’)
But what about this case?
←ATACGTAACGTATACCCGAC
TATATGCATTGCATATGGG→
5’3’
3’5’
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E. coli DNA polymerase has 3 domains:
One does the replication
One digests DNA 3’ → 5’ (exonuclease).
One digests DNA 5’ → 3’ (exonuclease).
Klenow fragment = engineered polymerase without the 5’ → 3’ exonuclease activity.
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ACGTAACGTATACCCGAC ATTGCATATGGGCTGAACAT
3’
5’
5’
3’
Polymerases always make the chain grow from the 5’ towards the 3’ (5’ → 3’)
But what about this case? Klenow has 3’ → 5’ exonuclease activity
←ATACGTAACGTATACCCGAC
TATATGCATTGCATATGGG→
5’3’
3’5’
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GAGGAGACGAACACCCGTATACAGTCGACG
GTATACAGTCGACGTTTATATATAACCCCGAGGAGACGA
The pieces are inserted into a vector – e.g., a plasmid. Sequencing is done from both sides
2. Sequence each piece:
One can use the same primers for all the sequencing. Parallelism of sequencing.
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GAGGAGACGAACACCCGTATACAGTCGACG
ACCCCGAGGAGACGA ? GTATACAGTCGACGTTTATATATA
GTATACAGTCGACGTTTATATATA
ACCCCGAGGAGACGA
Shotgun sequencing – why isn’t it a trivial task?
1. By chance, some parts are not sequenced even once!!!
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Shotgun sequencing – Definition of coverage.
X5 coverage: each base in the final sequence was present, on average, in 5 reads
Although the human genome was sequenced at a X12 coverage, still 1% of the genome is either not assembled or not reliable.
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Shotgun sequencing – why isn’t it a trivial task?
2. Some pieces do not align because of sequencing errors
GAGGTGAGGAACACCCGTATACAGTCGACG
ACCCCGAGG?GA?GAACACCCGTATACAGTCGACGTTTATATATA
ACCCCGAGGAGACGA
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Shotgun sequencing – why not a trivial task?
3. Repetitive sequences –satellites DNA.
GGGGGGGGGGGGGGGGGGGGGGGGGGGG
ACCCCGGGGGGGGGGGGG????GGGGGGGGGGGGGA
GGGGGGGGGGGGGGGGGGGGGGA
ACCCCGGGGG
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Shotgun sequencing – why isn’t it a trivial task?
4. Repetitive sequences (duplicated regions).In the genome we have duplicated regions which have almost identical sequence.
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Shotgun sequencing – why isn’t it a trivial task?
5. Some fragments are not sequenced because once inserted to a bacterium, they are toxic.
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A section of the genome that could be reliably assembled.
A contig
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A contig
Lander-Waterman estimation of number of contigs w.r.t. genome coverage
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At 8X-10X coverage, ~5 contigs are expected -> some of the genome is expected to be un-sequenced.
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Scaffolding
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Vector (e.g., e. coli)
Cloned fragment of the genome (e.g., 10 KB)
When sequencing a large genome, often the inserts are very large (10KB). In such case, it is impossible to sequence the entire insert, and only the edges are sequenced.
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Short fragments from both ends are sequenced
Mate pairsA read
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The size of the insert is also recorded.
Mate pairsA read 10 KB
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Information from mate pairs is used to build a scaffold of the genome
A contig
A contig
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The human genome is the chimp genome with 99% accuracy.
Comparative assembly
If one sequences the chimp genome – the information from the human genome can aid in the assembly.
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If one offers you to sequence your genome at 99.9% accuracy – don’t take it even for 5$.
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Often, phages are used as cloning vectors in standard cloning experiments. For genomic sequencing, Bacterial Artificial Chromosomes (BACs) are often used.
These are based on theF plasmid – a large plasmid that is stably replicating in E. coli.
Over 300kb can be insertedin the plasmid.
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The idea is to first divide a big genome to overlapping regions, put each in a BAC, and then use shotgun method to sequence each BAC.
BAC
BAC-by-BAC Assemble of the Genome
Into BAC
Shotgun
Sequencing the edges
Assemble each BAC
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Pyrosequencing: sequencing at the speed of light
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Pyrosequencing: a relatively new technique (invented 1986) in which the sequence of a DNA is discovered by synthesizing its complementary strand (the "sequencing by synthesis" principle).
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•Gel free
•Nucleotides are label free
•Parallelism
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GTP + DNA(n) -> DNA(n+1) + PPi
Enzyme = polymerase
PPi -> ATP
Enzyme = ATP Sulfurylase
ATP -> light
Enzyme = luciferase
ATP -> AMP + 2PPi
Enzyme = Apyrase
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ACGTAACGTATACCCG
TGCATT?
Only if one adds G – there will be light!
AC
GT
AA
CG
TA
TA
CC
CG
TG
CA
TT
?
1. Add ATP -> no light2. Add CTP -> no light3. Add GTP -> light4. Add TTP -> no light5. Add ATP -> no light6. Add CTP -> light7. Add GTP -> no light8. Add TTP -> no light9. Add ATP -> light
GCA Sequence = GCA
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Each DNA fragment was amplified and attached to a bead separately (one bead to each fragment). Each bead was added to a fibre-optic well.
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A computer can read the light pattern from billions of wells simultaneously.
(Sequencing of a bacterial genome in 7h).
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Your chip analysis suggests
stress
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1.Today, medicine is based on episodic treatment.
2.First step that is currently taken place is the use of digital imaging and their analysis (e.g., optic fibers).
3.Next step: “Digital health” – medical data for a person will be shared by all doctors – no matter where you are.
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4. Clinical genomics: fast and accurate identification of pathogens
5. Clinical genomics: sequence (part) of the genome to gain insights into which drugs are efficient.
6. Predisposition analysis for diseases.
7. Towards “lifetime treatment”…
8. Less doctor intuition – more quantitative parameters and statistical analysis.
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Difference between humans:
• SNP – single nucleotide polymorphism
• CGH – copy number variation
• Chromatin
• Epigenetics
We want to link these differences to diseases.
Bioinformatics and medicine
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Genomics
Proteomics
Metabolomics
System biology
In-silico (in vitro, in vivo)
Protein Engineering
Synthetic biology
Post genomic era
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Human DNA = ~2 meters
300 x 109 cells
3.2 x 109 nucleotides