1. Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX
Methods, Automation, Applications
Graham Wiley
Roe Lab

2. A Brief History of Automated DNA Sequencing Instruments
100,000,000
454/Roche GS-FLX
A Brief History of Automated DNA Sequencing Instruments
454-GS20
64,000,000
ABI 3730
ABI 370/377
ABI 3700
2007

3. 454 GSFLX Sequencer Pico-scale sequencing reactions 2 Core Techniques:
Emulsion PCR
Pyrosequencing

4. Emulsion PCR Micro-reactors
Water-in-oil emulsion generates millions of micelles.
Each micelle contains all reagents/templates for a PCR reaction.
~10 Million individual PCR reactions in a single tube.

5. Emulsion PCR

6. Load Beads into 454 Plate 44 μm Load Enzyme Beads
Load beads into PicoTiterPlate
Centrifugation
44 μm

7. Pyrosequencing DNA Bead dTTP (1) Polymerase PP APS (2) Sulfurylase
Polymerase adds
nucleotide (dNTP)
(1)
Polymerase
A A T C G G C A T G C T A A A A G T C A T PP i
APS
Annealed Primer
(2)
Pyrophosphate
is released (PPi)
Sulfurylase
Luciferase
ATP
(3)
Sulfurylase creates ATP
from PPi and APS
Enzyme Bead
(5)
luciferin
(4)
CCD camera detects bursts of light
Luciferase hydrolyses ATP
to oxidize luciferin and
produce light
Light + oxy luciferin

8. Pyrosequencing Output

9. Base Calling via Flowgram
TTCTGCGAA

10. Types of Libraries 454/Roche Roe Lab Shotgun Paired-End Amplicon
Random 250+bp reads
Paired-End
25-50bp ends of a circularized DNA molecule
Amplicon
PCR product for SNP discovery
Roe Lab
Paired-End/Shotgun
Best of both worlds

11. 454 Shotgun Library Preparation Protocol Overview
Nebulization
DNA End Repair 3’ 5’
Adaptor Ligation (A&B) 3’ 5’ B A
DNA End Repair 3’ 5’ B A
Library Quantification
on Caliper

12. 454 Paired End/Shotgun DNA Preparation Protocol Overview
Shear to 2-4 Kbp fragments on the Hydroshear
Quantitate on Caliper AMS-90
DNA End Repair & Linker Ligation
Cleave the Terminal Linkers with EcoR1
Ligate to Circularized the DNA
Shear to ~500 bp fragments in the Nebulizer

13. 454 Paired End/Shotgun DNA Preparation Protocol Overview (cont)
Quantitate on Caliper AMS-90
DNA End Repair, Adaptor Ligation,
Adapter End Repair
Amplification (emPCR)
Pyrosequencing on 454/Roche GS-FLX

14. 454 Paired-End/Shotgun Separate Sequences Triple Assembly
Linker?
Left and Right?
~3-5%
Triple Assembly
100 flows, 84 flows, 62 flows
Convert Paired Ends for Exgap
*.454f and *.454r

15. Automation of the Library Preparation Steps
Why automate?
Time
Reproducibility
What are the obstacles?
Reaction Cleanup
Qiagen Minelute centrifuge columns are difficult to automate, so replace those steps with
Agencourt SPRI magnetic beads and add a magnetic station to the Zymark SciClone bed
Enzyme Stability and Storage
Build an enzyme cooling station on the Zymark SciClone bed

16. SPRI Bead Technology Solid Phase Reversible Immobilization
Solid Phase Reversible Immobilization
Carboxyl coated magnetic particles suspended in a solution of 10% PEG and 1.25M NaCl
Reversibly binds DNA
Hawkins, et al. (1994) DNA purification and isolation using a solid-phase. Nucleic Acids Research, 22(21):

17. DNA Purification through the Qiagen Minelute Columns vs Agencourt SPRI Magnetic Beads
Qiagen Minelute centrifuge column
Agencourt SPRI magnetic beads
Both procedures give an almost similar yield but the yield is slightly better with the SPRI beads and the automation of the SPRI bead prep is somewhat easier to achieve

18. 96 well Magnetic Plate for Purification of the SPRI Beads

19. Enzyme Chilling Station

20. Zymark SciClone Deck Arrangement
Waste
EtOH
Magnet
Enzyme Mixes
Shaker
Shaker
Sample
Buffers
Shaker
SPRI Beads

21. Adding SPRI Beads on the SciClone

22. Magnetically Separating SPRI Beads on the SciClone

23. Washing SPRI Beads on the SciClone

24. Applications Whole Genome Sequencing Sample Pools EST Libraries
BACs
Viruses
EST Libraries
Bacterial Communities

25. Xanthamonas campestris
Optimization of the DNA to Bead Ratios used in emPCR???
Xanthamonas campestris
~5.0 MB genome
47.9 MB total sequence
~10 x coverage

26. Xanthamonas campestris

27. Heterosigma akashiwo Virus
Kb Genome
13Mb Sequenced
~ 43x Coverage
Contig Size Total Number Total Length % Of Cumulative
0 - 1 kb %
1 - 2 kb %
2 - 3 kb %
3 - 4 kb %
4 - 5 kb %
kb %
kb %
kb %
kb %
kb %
kb %
>100 kb %
Cumulative Consed_Err/10KB =
Cumulative>1 kb Consed_Err/10KB = 213.7
Cumulative>2 kb Consed_Err/10KB = 30.07

28. Current BAC Pooling Strategy
10x10 Grid of 100 BAC clones
1-fold coverage of each pool of Kb BACs is 1.5 Mb
1 quarter 454/Roche GS-FLX picotiter plate give ~13Mb or 10-fold cov.
5 full picotiter plate runs are required for 20-fold coverage of each individual BAC at the horizontal/vertical intersect.
$12k/run = ~$600/BAC
Additional ABI 3730 runs are needed for each pool to aid in deconvolution at ~ $1000 for each of the 20 pools and an additional ~800/BAC or $1400 total cost per BAC
Pool B
Pool 3 X

29. Future Tagged BAC Pooling Strategy
24 uniquely tagged individual shotgun libraries would be pooled and sequenced on one full 454/Roche GS-FLX picotiter plate
Kb BACs would require 3.6 Mb for 1 x sequence coverage
With >75 Mb of DNA sequence obtained per full plate, >20x coverage is obtained for each of the 24 pooled BACs
96 BACs would therefore require 4 full plate runs on the 454/Roche GS-FLX
At $12k/run = ~$500 per BAC for >20-fold shotgun coverage and no ABI 3630 runs are needed to deconvolute the individual BACs as each BAC is individually tagged

30. Conclusions Graham- these are not conclusions, conclusions are of the form, we observed this and it means the following!!
New techniques lead to more useful data
It is possible to automate 454 preparation protocols
454 pyrosequencing technology is incredibly powerful
Massive amounts of data
Technique advances continue to lower costs

31. Acknowledgments

32. Qiagen Minelute vs Agencourt SPRI
50ul nebulized DNA
20ul control
15ul through Qiagen Minelute centrifuge column (final volume 25ul)
15ul through Agencourt SPRI beads (final volume 25ul)
Control