DNA colony massively parrallel sequencing ams98 presentation

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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).

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DNA colony massively parrallel sequencing ams98 presentation

  1. 1. A Very Large Scale, High Throughput and Low CostDNA Sequencing Method based on a New2-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. 2. 3’ end free in solution 5~10nmPrimers 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. 3. Anneal Elongate Separate strands wash < d > = f ( [PS] )INITIATECycle 1 h < 300 nmCycle 2 ... < s > = f ( number cycles )Cycle n => DNA colonies 28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.3/8
  4. 4. Experimental conditions Prepared T1 samples : T2Support : NucleolinkTM tubes covered with red and greenoligos 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. 5. DNA colonies 2~10 µm 1~2µmsamples : 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. 6. Sequencing : stepwise primer extensionusing DNA polymerase Fluorescence increaseand fluorescent nucleotides on individual DNA colonies A AAPrimerTemplate A A A Wash + Image analysis AC AACWash +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. 7. 222 304 215 264 394 212+CA B +T ..TGACT+CC 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. 8. Extrapolating from current experimental results :EXPECTED THROUGHPUTUltimate 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 -> x20longer term : 1s exposure -> x200) 28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.8/8
  9. 9. 17 bases28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.9/8
  10. 10. Pharmacogenomics: relating genotype to drug responseMany polymorphism... in many genes... in many patients !!! => Giga base projects 28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.10/8
  11. 11. Assay monitoringSamplecollection Sequence analysis Sample assaying Sample preparation & arraying28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.11/8
  12. 12. Assay monitoringGenomic 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

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