1

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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).

2: Large-Scale37 / 42 Bioinformatics and medicine

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

2: Large-Scale42 / 42 Some important NUMBERS

Human DNA = ~2 meters

300 x 109 cells

3.2 x 109 nucleotides