2: Large-Scale

Large!

2: Large-Scale

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

Genomic Sequencing – shotgun sequencing

Sequencing is usually ~700 bp in a single run.

How can we sequence a genome?

2: Large-Scale

2: Large-Scale

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

2: Large-Scale

GAGGAGACGAACACCCGTATACAGTCGACG

ACCCCGAGGAGACGAACACCCGTATACAGTCGACGTTTATATATA

GTATACAGTCGACGTTTATATATA

ACCCCGAGGAGACGA

Genomic Sequencing – shotgun sequencing

1. Break DNA to small pieces2. Sequence each piece3. Assemble

2: Large-Scale

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

2: Large-Scale

<-ATACGTAACGTATACCCGACTATATGCATTGCATATGGG->

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’)

2: Large-Scale

ACGTAACGTATACCCGAC ATTGCATATGGGCTGAACAT

3’

5’

5’

3’

<-ATACGTAACGTATACCCGACTATATGCATTGCATATGGG->

3’

5’

5’

3’

Polymerases always make the chain grow from the 5’ towards the 3’ (5’->3’)

But what about this case?

2: Large-Scale

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.

2: Large-Scale

ACGTAACGTATACCCGAC ATTGCATATGGGCTGAACAT

3’

5’

5’

3’

<-ATACGTAACGTATACCCGACTATATGCATTGCATATGGG->

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

2: Large-Scale

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.

2: Large-Scale

GAGGAGACGAACACCCGTATACAGTCGACG

ACCCCGAGGAGACGA ? GTATACAGTCGACGTTTATATATA

GTATACAGTCGACGTTTATATATA

ACCCCGAGGAGACGA

Shotgun sequencing – why isn’t it a trivial task?

1. By chance, some parts are not sequenced even once!!!

2: Large-Scale

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.

Shotgun sequencing – why isn’t it a trivial task?

2. Some pieces do not align because of sequencing errors

2: Large-Scale

GAGGTGAGGAACACCCGTATACAGTCGACG

ACCCCGAGG?GA?GAACACCCGTATACAGTCGACGTTTATATATA

ACCCCGAGGAGACGA

Shotgun sequencing – why not a trivial task?

3. Repetitive sequences –satellites DNA.

2: Large-Scale

GGGGGGGGGGGGGGGGGGGGGGGGGGGG

ACCCCGGGGGGGGGGGGG????GGGGGGGGGGGGGA

GGGGGGGGGGGGGGGGGGGGGGA

ACCCCGGGGG

2: Large-Scale

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.

2: Large-Scale

Shotgun sequencing – why isn’t it a trivial task?

5. Some fragments are not sequenced because once inserted to a bacterium, they are toxic.

2: Large-Scale

A section of the genome that could be reliably assembled.

A contig

2: Large-Scale

A contig

Lander-Waterman estimation of number of contigs w.r.t. genome coverage

2: Large-Scale

At 8X-10X coverage, ~5 contigs are expected -> some of the genome is expected to be un-sequenced.

2: Large-Scale

Scaffolding

2: Large-Scale

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.

Short fragments from both ends are sequenced

2: Large-Scale

Mate pairsA read

The size of the insert is also recorded.

2: Large-Scale

Mate pairsA read 10 KB

2: Large-Scale

Information from mate pairs is used to build a scaffold of the genome

A contig

A contig

2: Large-Scale

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.

2: Large-Scale

If one offers you to sequence your genome at 99.9% accuracy – don’t take it even for 5$.

2: Large-Scale

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 the F plasmid – a large plasmid that is stably replicating in E. coli.

Over 300kb can be inserted in the plasmid.

2: Large-Scale

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

2: Large-Scale

Pyrosequencing: sequencing at the speed of light

2: Large-Scale

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).

2: Large-ScalePyrosequencing:

•Gel free

•Nucleotides are label free

•Parallelism

2: Large-Scale

•GTP + DNA(n) -> DNA(n+1) + PPi

Enzyme = polymerase

•PPi -> ATP

Enzyme = Sulfurylase

ATP -> light

Enzyme = luciferase

ATP -> AMP + 2PPi

Enzyme = Apyrase

2: Large-ScalePyrosequencing

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

2: Large-ScalePyrosequencing

Each DNA fragment was amplified and attached to a bead seperately (one bead to each fragment). Each bead was added to a fibre-optic well.

2: Large-ScalePyrosequencing

A computer can read the light pattern from billions of wells simultaneously.

(Sequencing of a bacterial genome in 7h).

2: Large-ScaleBioinformatics and medicine

Your chip analysis suggests stress

2: Large-ScaleBioinformatics and medicine

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.

2: Large-Scale Bioinformatics and medicine

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. To relay less on the intuition of the doctor – more on quantitative parameters and statistical analysis.

2: Large-Scale

Difference between humans:

• SNP – single nucleotide polymorphism

• CGH – copy number variation

• Chromatin

• Epigenetics

We want to link these differences to diseases.

Bioinformatics and medicine

2: Large-ScaleSome more important buzz words

Genomics

Proteomics

Metabolomics

System biology

In-silico (in vitro, in vivo)

Protein Engineering

Synthetic biology

Post genomic era

2: Large-ScaleSome important NUMBERS

Human DNA = ~2 meters

300 x 109 cells

3.2 x 109 nucleotides

2: Large-Scale

Chip arrays and gene expression data

2: Large-Scale

With the chip array technology, one can measure the expression of 10,000 (~all) genes at once. Can answer questions such as:

1.Which genes are expressed in a muscle cell?

2.Which genes are expressed during the first weak of pregnancy in the mother? In the new baby?

3.Which genes are expressed in cancer?

2: Large-Scale

4. If one mutates a TF: which genes are not expressed following this change?

5. Which genes are not expressed in the brain of a retarded baby?

6. Which genes are expressed when one is asleep versus when the same person is awake?

2: Large-Scale

DNA chip: in each cell there’s a specific DNA molecule. Upon hybridization with an mRNA molecule (or a cDNA one) – the intensity of the hybridization can be quantified by light.

2: Large-Scale

Affymetrix: The base is a “wafer” מצע גבישי מוליך למחצה דק

A light-sensitive chemical compound that prevents coupling between the wafer and the first nucleotide of the DNA probe being created.

2: Large-Scale

The blue “cap” is light sensitive. A mask is added to some of the cells. When the cells are illuminated, only where there is light – a reaction with the nucleotide can happen.

Affymetrix

2: Large-Scale

The nucleotide that is added is also chemically linked with a new “cap” (light sensitive).

Affymetrix

2: Large-Scale

The entire process is called photolithography

Affymetrix

2: Large-Scale

Affymetrix: each probe is 25 bp – a part of an exon.

The readerThe chip itself

In one cm2 > 106 different oligos.

Affymetrix

2: Large-Scale

Affymetrix: each probe is 25 nucleotides. Above this, a technological problem exists: the synthesis becomes inaccurate.

With such short probes, each mRNA can hybridize to more than one probe. The solution, each gene is “covered” by several probes.

Affymetrix

2: Large-Scale

Affymetrix: one can buy ready-made chips (human genome, mouse genome), or can design (“print”) his own chip (more expensive).

Affymetrix

2: Large-Scale

Detection: mRNA is isolated from the tissue (cells, viruses). cDNA is synthesized. The cDNA is fluorescently labeled. Sometimes, the cDNA is amplified using PCR. The intensity in each cell (probe) is measured by “the reader”.

Affymetrix

2: Large-Scale

AgilentDeveloped DNA printers – in each spot pico-liters of nucleotides are added. They can make probes up to 60 mers (Agilent is derived from Hewlett-Packard).

Agilent

Standard phosphoramidite chemistry

2: Large-Scale

AgilentHybridization to Agilent probes is more accurate.

If there is hybridization to a probe, the gene it represents is probably expressed.

Agilent

2: Large-Scale

But, it is impossible to know how many probes are in each cell. So absolute fluorescent intensities are meaningless.

Agilent

2: Large-Scale

Solution, in the same experiment, hybridize samples with two conditions: healthy mRNA (in Red) versus tumor cells (green).

The Agilent reader will give the ratio of the two colors.

Agilent

2: Large-Scale

In this approach, long cDNA sequences (>300bp) are produced in a cell (a clone) and are linked to each chip cell. Producing long cDNA rather than synthesizing them a nucleotide at a time is cheaper!

As in the case of Agilent, it is impossible to control the number of probes in each cell.

Stanford cDNA chips

2: Large-Scale Output

w.tBrain tumor

males

Brain tumor

females

Gene 1

Gene 2

Gene 3

Gene 60000

Each cell is either an absolute number or a relative one, depending on the technology used.

2: Large-Scale Repeats

w.tBrain tumor

male1

Brain tumor

male2

Brain tumor

female1

Gene 1

Gene 2

Gene 3

Gene 60000

The repeat can either be the same sample – a different chip or a “real” biological repeat – a different sample.

2: Large-ScaleExpression profile

wt1wt2wt3wt4bt1bt2bt3bt4

g1435415161723

g275466379

g3232525263060

Genes 1 and 3 show the same trend (go both high under the same conditions). That is: they have the same expression profile.

2: Large-ScaleClustering

wt1

wt2

wt3wt4bt1bt2bt3bt4

g1435415161723

g275466379

g3232525263060

In general, we want to find all the genes which share the same expression profile -> suggestive of a functional linkage.

This is done by clustering genes with the same profile

2: Large-ScaleClustering

wt1

wt2

wt3wt4bt1bt2bt3bt4

g14354022023

g275460809

g32325601661

Clustering of the conditions can suggest two types of brain tumor (bt)

Bi-clustering: both on the conditions and the genes.

2: Large-ScaleApplications

Think of increasing the glucose concentration of E.coli and making a chip array in various concentrations.

One can potentially discover all genes in the glucose pathway.

Knocking out a gene -> discover all genes that interact with it.

2: Large-ScaleApplications

Analyzing expression of genes can help reveal the gene network of a given organism.

2: Large-ScaleGene network

2: Large-ScaleClinical

Tal

g111

g24

g30

Do I have a brain tumor?

wt1

wt2

wt3wt4bt1bt2bt3bt4

g14354022023

g275460809

g32325601661

2: Large-Scale Sequence by hybridizationIt was thought that the following procedure could work for sequencing a genome:

1.Make a chip containing all x mers (e.g., x = 25).2.Hybridize a genome to the chip.3.By analyzing all the hybridizations with their overlaps – assemble the genome.

Problem: it doesn’t work.

2: Large-Scale

ChIP-chip