First public presentation of massively parallel sequencing proof of principle (DNA colony base-by-base sequencing).
“A very large scale, high throughput and low cost DNA sequencing method based on a new 2-dimensional DNA auto-patterning process”,P. Mayer, L. Farinelli, G. Matton, C. Adessi, G. Turcatti, J.J. Mermod, E. Kawashima, presented at the Fith International Automation in Mapping and DNA Sequencing Conference, St. Louis (MI,USA), October 7-10, 1998. Invited presentation (P. Mayer).
Decarbonising Buildings: Making a net-zero built environment a reality
DNA colony massively parrallel sequencing ams98 presentation
1. A Very Large Scale, High Throughput
and Low Cost
DNA Sequencing Method based on a New
2-Dimensional DNA Auto-Patterning Process
P. Mayer, (L. Farinelli), G. Matton, C. Adessi, G.
Turcatti, J.J. Mermod, E. Kawashima.
Genomic Technology Department
Serono Pharmaceutical Research Institute
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.1/8
2. 3’ end free in solution
5~10nm
Primers p1
+ p2
Covalent attachment
0.3 ~ 2 cm
Prepared sample 1
Prepared sample 2
.
. ~1kb => ~300nm > persistence length
.
Prepared sample n
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.2/8
3. Anneal Elongate Separate strands
wash
< d > = f ( [PS] )
INITIATE
Cycle 1
h < 300 nm
Cycle 2
...
< s > = f ( number cycles )
Cycle n
=> DNA colonies
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.3/8
4. Experimental conditions
Prepared T1
samples :
T2
Support : NucleolinkTM tubes covered with red and green
oligos
3mm
Colony formation with different mixes of T1 and T2
Probe with biotinylated nick-translation DNA probes and
40x
fluorescent streptavidin-beads (40nm)
Setup : inverted epi-fluorescence microscope with 40x objective,
cooled CCD camera, computer
CCD
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.4/8
5. DNA
colonies
2~10 µm 1~2µm
samples : 100% T1, 0% T2 samples : 0% T1,100% T2
10µm 10µm
Hybridization with probe
specific to T1
=> Number of spots ∝ samples
=> 1~10 million samples / cm2
samples 10% T1,90% T2 10µm
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.5/8
6. Sequencing : stepwise primer extension
using DNA polymerase
Fluorescence increase
and fluorescent nucleotides on individual DNA colonies
A
AA
Primer
Template
A A A
Wash + Image analysis
AC
AAC
Wash +
Image analysis C
C n cycles T
Wash + Image AACG
analysis
AG CG
Wash + Image analysis
G
in average, 2 bases read / cycle
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.6/8
7. 222 304 215 264 394 212
+C
A B
+T
..TGACT
+C
C seq 1 : TATACTGACCT Cy5 labeled
..TGAT
seq 2 : GCTACTATTCT
267 383 217 Objectives :
~10 bases = tag counting, genotyping
>20 bases = “sequencing”
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.7/8
8. Extrapolating from current experimental results :
EXPECTED THROUGHPUT
Ultimate limiting factors, presently : time to acquire an image, ~10s
colony density, 10,000 colonies/image (20x objective, 2kx2k CCD)
raw average => 5000 bases/10s -> usable :
50~500 bases/s (“ABI” 1~4 base/s)
EXPECTED COSTS :
~$1000 for 2 day operation on 18x18 mm “chips”
-> 6.106-7 bases raw sequence
=> $1 = 6,000~60,000 bases (“ABI” 3~10 base/$)
(optimistic : 10x objective, 5.104 colonies/image -> x20
longer term : 1s exposure -> x200)
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.8/8
9. 17 bases
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.9/8
10. Pharmacogenomics:
relating genotype to drug response
Many polymorphism...
in many genes...
in many patients !!!
=> Giga base projects
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.10/8
11. Assay monitoring
Sample
collection
Sequence
analysis
Sample
assaying
Sample
preparation & arraying
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.11/8
12. Assay monitoring
Genomic Technology Dpt. :
“Nano/micro technology”
based on
Sample biochemical auto-patterning
assaying
avoiding (micro-)robotics
& micro-lithography
Sample
preparation & arraying
28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.12/8