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 Assembly Process
Separate based on inclusion or exclusion of middle linker
Those sequences containing a middle linker are further separated based on the length of the read to either end of the linker sequence
~3-5% of the total reads contain the middle linker sequence
Assembly of the reads by Newbler
Convert paired ends for Exgap ordering and orienting
*.454f and *.454r

15. Automation of the Shotgun 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. Plant Viruses of the Tallgrass Prairie
Single or double stranded RNA
Typically <10,000bp, ~12,000bp max.
4-12 encoded genes
Inherent instability of RNA leads to large amount of mutations, hence, large species variation

26. cDNA pooling strategy
Tags on PCR primers allow for deconvolution of viral sequences post sequencing
cDNA samples are pooled in sets of at the Noble Foundation and sent to OU for sequencing

27. Strategy for preparing cDNA ready for 454 sequencing from dsRNA
5’ 3’
3’ 5’
Anneal with Random Hexamer Primers followed by Reverse Transcriptase PCR Reaction 5’ 5’ 3’
CCTCCTAGGCTTCC
NNNNNN
NNNNNN
CCTTCGGATCCTCC + 5’ 3’ 5’ 5’
Additional Rounds of RT PCR with Random Hexamer Primers
NNNNNN
CCTTCGGATCCTCC
3’ 5’
CCTCCTAGGCTTCC
NNNNNN +
5’ 3’
CCTTCGGATCCTCC
NNNNNN
CCTCCTAGGCTTCC
NNNNNN
RNAse Treatment to Remove any Excess Random Hexamer Primers followed by a Taq Polymerase PCR with one of the 20 Tagged Primers
5’ 3’
3’ 5’
CCTTCGGATCCTCC
NNNNNN
GGAAGCCTAGGAGG
CCTCCTAGGCTTCCGAGA +
3’ 5’
5’ 3’
GGAAGCCTAGGAGG
NNNNNN
CCTCCTAGGCTTCC
AGAGCCTTCGGATCCTCC
Amplified Product Ready for Ligating 454 A and B Primers 5’ A
AGAGCCTTCGGATCCTCC B
CCTCCTAGGCTTCCGAGA

28. Uniquely Tagged cDNA Sample from the TGP on the 454
454 tag (TCAG)
TGP Unique tag (GACA)
TGP common primer
(CCTTCGGATCCTCC)
RT-PCR Sequence

29. Putative New Allexivirus
Membrane
Protein
Nucleic Acid
Binding Protein
(+)ssRNA
Hypothetical
Protein
Coat
Protein
Replicase
Helicase
~8.5KB 13 76 65 74 69
105 75 81 95 83
05TGP00120
BlastX shows a large number of contigs have homology to viruses of the genus Allexiviridae
Contig sequence lengths cover ~66% of a typical Allexivirus genome of ~8.5KB
5 of the 6 genes encoded by Allexiviridae species are represented in the sequenced contigs

30. 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 A X

31. 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 3730 runs are needed to deconvolute the individual BACs as each BAC is individually tagged

32. Conclusions
It is possible to incorporate both shotgun and paired end reads in the same library
Qiagen Minelute centrifuge columns may be replaced by Agencourt SPRI beads after enzymatic steps in 454 library preparation.
The replacement of centrifuge columns with magnetic beads as well as the manufacture of an enzyme chilling station allows for the automation of the library making process
Through the use of tagged RT-PCR samples it is possible to sequence putatively novel plant viruses

33. Acknowledgments Dr. Roe
Loaders & Data Analyzers: Simone Macmil, Doug White, Steve Kenton
Makers & Breakers: Chunmei Qu, Ping Wang, Yanbo Xing, Baifeng Qin, Keqin Wang
All other members of the Roe lab
Collaborators
OSU: Ulrich Melcher, Vijay Muthamukar
Noble Foundation: Marilyn Roossinck, Guoan Shen, Byoung Min, Rick Nelson, Tracy Feldman