Arrays against time
Transcriptomics
‘101’
Wuhan 2011 CCC
Other species
Over expressed
WT
Mutant
Transcription assay: Northerns
Label probe
+ hybridise
quantitate
Extract target
RNA
YFG
Next
gene
Problems with Northerns:
• Slow (Time consuming)
• Hard (Technically challenging)
•
捡了芝麻丢了西瓜
Systems biology networks
- We want to look at lots of transcripts:
Aracyc +other metabolomics data
Arabidopsis gene network
(Ma et al. Genome Research 2007)
Arabidopsis
AtRegNet (gene regulation network)
Merged Network
Proteins (red) Metabolites (blue) & Genes (green)
19392 nodes and 72715 edges
捡了芝麻丢了西瓜
Other species
Over expressed
WT
Mutant
Northerns – a few genes at a time.
Label probe
+ hybridise
YFG
Again and again and….
quantitate
Extract target
RNA
Next
gene
Mass transcript profiling: Transcriptomics
• Sequencing ESTs
(Déjà vu?)
• Differential display
(random 5’ primers
+ fixed polyA primers)
• Microarrays
Probe preparation
Target preparation
Acquire or
Generate probes
‘All the genes you want’
Extract RNA from your
Control AND your
Experimental plant
Label cDNA
from sample 1 RNA
…and sample 2 RNA
Spot
Hybridise & Scan
Identify ‘spots’
remove background
produce ‘red/green’ ratios
• Link ratio to relative abundance.
• Link spot to gene.
• Link genes to each other.
Arrays
How do you make them ?
Arrayers
Pins
Pin type: blunt, ring, quill, coated…..
Breaking: bending, sticking
Consistency of spots: ‘coffee-cup’, splash, drip
Contamination: carry-over, dust, hairs, crystals.
Etc etc….
Slides
• Cracking
• Splitting
• Exfoliating
• Fluorescing
• Coatings - Hydrophobic, hydrophilic, correctly aged polylysine (a bit of an art)
• Home-made vs bought (cost of internal vs external quality
control.
• Scan before coating, scan after coating, scan after arraying,
scan after hyb-ing all part of QC
•Etc…etc...
The finished spotted array
Before processing, we have a LOT of spots
Example Hybridisation
After processing, we have a LOT of objective data
What biological questions
can you answer with arrays ?
Sorting out gene families
5 hormone response
gene family members
In different experiments
1. +hormone vs ctrl hyb
2. Normal vs mutant hyb
3. Root vs shoot hyb
microarray
What goes on the slide ?
The original choice was:
Mass amplifications of cDNAs
identified by partial sequence
(ESTs)
However …..
Duplication in genomes
is a real problem
Human
Plant
Yeast
Apart from wholesale duplication
Gene families:
(# of members as a proportion of the genome)
Unique
35%
2
12.5%
3
7%
4
4.4%
5
3.6%
Conservation between genes:
• 37% of genes are highly conserved
(TBLASTX E<10-30)
• 10% more are partially conserved
(TBLASTX E<10-5)
>5
37.4%
ESTs have inherent problems
Gene of interest
Homologous EST sequence
1
2
Dissimilar EST sequence
3
Example EST sequence
On the slide
1
2
3
Labelled target may hybridise similarly to each
Better solutions:
• GSTs (gene specific tags)
• Oligo arrays
• Affymetrix genechips
• RNA seq???
Selection of
Expression Probes
5’
3’
Sequence
Probes
Perfect Match
Mismatch
Chip
Affymetrix
Wafer and Chip Format
5 - 50 µm
5 - 50 µm
Millions of identical
oligonucleotide
probes per feature
49 - 400
chips/wafer
1.28cm
up to ~ 3,000,000 features/chip
Probe cells of an Affymetrix GeneChip
contain millions of identical 25-mers
25-mer
Photolithographic Synthesis
Lamp
Mask
Chip
Synthesis of Ordered Oligonucleotide Arrays
One nucleotide at a time.
here
Procedures for Target Preparation
AAAA
RNA
RNA Quality control
Procedures for Target Preparation
B
Biotin-labeled
transcripts
B
B
B
B
Fragment
(heat, Mg2+)
B
B
B
Fragmented cRNA
IVT
AAAA
RNA
(Biotin-UTP
Biotin-CTP)
Wash & Stain
Scan
cDNA
Hybridise
(16 hours)
GeneChip® Expression Analysis
Hybridization and Staining
Array
Hybridized Array
cRNA Target
Ab detection
Affymetrix software derives the intensity for each
probe from the 75% quantile of the pixel values in
each box.
Expression Measure
The intensities of the multiple probes within a probeset
are combined into ONE measure of expression
Chips need to be normalised against each other.
Each chip is a
different colour
in this graph
They are not
co-incident for
intensities
To compare
they need to
be comparable
PA
PB
PC
PD
PE
Chip 1
1
2
4
3
5
Chip 2
7
2
5
3
1
Chip 3
5
3
4
2
9
Chip 1
1
2
3
4
5
Chip 2
1
2
3
5
7
Chip 3
2
3
4
5
9
Order by ranks
Average the intensities at each rank
Chip 1
1.33
2.33
3.33
4.66
7
Chip 2
1.33
2.33
3.33
4.66
7
Chip 3
1.33
2.33
3.33
4.66
7
PA
PB
PC
PD
PE
1.33 2.33
4.66
3.33
7
2.33
4.66
3.33
1.33
4.66 2.33
3.33
1.33
7
Reorder by probe
Chip 1
Chip 2
Chip 3
7
RMA uses
normalisation
at the probe
level
R/
BioConductor
training
Normalisation, filtering and annotation
AffylmGUI
training
.CDF , filtering, stats and annotation
Xspecies
analysis
training
RMA Normalisation
Sequencing: current / next gen / future
Sequencing is likely to complement arrays in the future
Standard (Sanger) sequencing
Random
Primer
P
ddNTP termination.
r
i
m
Template
e
r
Label can be added to the:
• Primer
• ddNTP –or• Incorporated dNTPs
454 sequencing (images by Roche)
Sample Input and Fragmentation: Genomic DNA or BACs are
fractionated into small, 300- to 800-basepair fragments
Library Preparation: Short adaptors (A and B) - specific for both
the 3' and 5' ends - are added to each single stranded fragment.
One Fragment = One Bead:
Each fragment of the single-stranded DNA library is immobilized
individually onto beads in a water-in-oil mixture.
emPCR (Emulsion PCR) Amplification:
Each unique fragment is amplified in parallel to several million per
bead.
One Bead = One Read: The clonally amplified fragments are
loaded onto a PicoTiterPlate device for sequencing.
Only one bead per well.
Auto fluidics flows individual nucleotides in a fixed order across
the hundreds of thousands of wells containing one bead.
Addition of a nucleotide results in a chemiluminescent signal.
Solexa sequencing I
Series of images taken from www.illumina.com
Solexa sequencing II
Solexa sequencing III
But the future may be even
faster……
• http://www.pacificbiosciences.com/aboutus/video-gallery
•
Note: Direct link may be disallowed by the server.
– try direct paste into a browser and click the SMRT Biology Overview in the video-gallery archive
Rubber sequencing