2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression...
-
date post
21-Dec-2015 -
Category
Documents
-
view
225 -
download
2
Transcript of 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression...
![Page 1: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/1.jpg)
2: Large-Scale
Large!
![Page 2: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/2.jpg)
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
![Page 3: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/3.jpg)
Genomic Sequencing – shotgun sequencing
Sequencing is usually ~700 bp in a single run.
How can we sequence a genome?
2: Large-Scale
![Page 4: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/4.jpg)
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
![Page 5: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/5.jpg)
2: Large-Scale
GAGGAGACGAACACCCGTATACAGTCGACG
ACCCCGAGGAGACGAACACCCGTATACAGTCGACGTTTATATATA
GTATACAGTCGACGTTTATATATA
ACCCCGAGGAGACGA
Genomic Sequencing – shotgun sequencing
1. Break DNA to small pieces2. Sequence each piece3. Assemble
![Page 6: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/6.jpg)
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
![Page 7: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/7.jpg)
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’)
![Page 8: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/8.jpg)
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?
![Page 9: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/9.jpg)
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.
![Page 10: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/10.jpg)
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
![Page 11: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/11.jpg)
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.
![Page 12: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/12.jpg)
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!!!
![Page 13: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/13.jpg)
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.
![Page 14: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/14.jpg)
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
![Page 15: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/15.jpg)
Shotgun sequencing – why not a trivial task?
3. Repetitive sequences –satellites DNA.
2: Large-Scale
GGGGGGGGGGGGGGGGGGGGGGGGGGGG
ACCCCGGGGGGGGGGGGG????GGGGGGGGGGGGGA
GGGGGGGGGGGGGGGGGGGGGGA
ACCCCGGGGG
![Page 16: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/16.jpg)
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.
![Page 17: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/17.jpg)
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.
![Page 18: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/18.jpg)
2: Large-Scale
A section of the genome that could be reliably assembled.
A contig
![Page 19: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/19.jpg)
2: Large-Scale
A contig
Lander-Waterman estimation of number of contigs w.r.t. genome coverage
![Page 20: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/20.jpg)
2: Large-Scale
At 8X-10X coverage, ~5 contigs are expected -> some of the genome is expected to be un-sequenced.
![Page 21: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/21.jpg)
2: Large-Scale
Scaffolding
![Page 22: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/22.jpg)
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.
![Page 23: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/23.jpg)
Short fragments from both ends are sequenced
2: Large-Scale
Mate pairsA read
![Page 24: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/24.jpg)
The size of the insert is also recorded.
2: Large-Scale
Mate pairsA read 10 KB
![Page 25: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/25.jpg)
2: Large-Scale
Information from mate pairs is used to build a scaffold of the genome
A contig
A contig
![Page 26: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/26.jpg)
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.
![Page 27: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/27.jpg)
2: Large-Scale
If one offers you to sequence your genome at 99.9% accuracy – don’t take it even for 5$.
![Page 28: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/28.jpg)
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.
![Page 29: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/29.jpg)
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
![Page 30: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/30.jpg)
2: Large-Scale
Pyrosequencing: sequencing at the speed of light
![Page 31: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/31.jpg)
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).
![Page 32: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/32.jpg)
2: Large-ScalePyrosequencing:
•Gel free
•Nucleotides are label free
•Parallelism
![Page 33: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/33.jpg)
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
![Page 34: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/34.jpg)
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
![Page 35: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/35.jpg)
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.
![Page 36: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/36.jpg)
2: Large-ScalePyrosequencing
A computer can read the light pattern from billions of wells simultaneously.
(Sequencing of a bacterial genome in 7h).
![Page 37: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/37.jpg)
2: Large-ScaleBioinformatics and medicine
Your chip analysis suggests stress
![Page 38: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/38.jpg)
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.
![Page 39: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/39.jpg)
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.
![Page 40: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/40.jpg)
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
![Page 41: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/41.jpg)
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
![Page 42: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/42.jpg)
2: Large-ScaleSome important NUMBERS
Human DNA = ~2 meters
300 x 109 cells
3.2 x 109 nucleotides
![Page 43: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/43.jpg)
2: Large-Scale
Chip arrays and gene expression data
![Page 44: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/44.jpg)
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?
![Page 45: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/45.jpg)
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?
![Page 46: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/46.jpg)
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.
![Page 47: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/47.jpg)
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.
![Page 48: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/48.jpg)
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
![Page 49: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/49.jpg)
2: Large-Scale
The nucleotide that is added is also chemically linked with a new “cap” (light sensitive).
Affymetrix
![Page 50: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/50.jpg)
2: Large-Scale
The entire process is called photolithography
Affymetrix
![Page 51: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/51.jpg)
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
![Page 52: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/52.jpg)
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
![Page 53: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/53.jpg)
2: Large-Scale
Affymetrix: one can buy ready-made chips (human genome, mouse genome), or can design (“print”) his own chip (more expensive).
Affymetrix
![Page 54: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/54.jpg)
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
![Page 55: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/55.jpg)
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
![Page 56: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/56.jpg)
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
![Page 57: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/57.jpg)
2: Large-Scale
But, it is impossible to know how many probes are in each cell. So absolute fluorescent intensities are meaningless.
Agilent
![Page 58: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/58.jpg)
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
![Page 59: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/59.jpg)
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
![Page 60: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/60.jpg)
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.
![Page 61: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/61.jpg)
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.
![Page 62: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/62.jpg)
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.
![Page 63: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/63.jpg)
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
![Page 64: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/64.jpg)
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.
![Page 65: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/65.jpg)
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.
![Page 66: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/66.jpg)
2: Large-ScaleApplications
Analyzing expression of genes can help reveal the gene network of a given organism.
![Page 67: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/67.jpg)
2: Large-ScaleGene network
![Page 68: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/68.jpg)
2: Large-ScaleClinical
Tal
g111
g24
g30
Do I have a brain tumor?
wt1
wt2
wt3wt4bt1bt2bt3bt4
g14354022023
g275460809
g32325601661
![Page 69: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/69.jpg)
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.
![Page 70: 2: Large-Scale Large!. 2: Large-Scale High throughput technologies: Sequencing Gene expression profiling Chip-CHIP and tiling arrays Whole genome yeast.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d6c5503460f94a4bd66/html5/thumbnails/70.jpg)
2: Large-Scale
ChIP-chip