1. Massively Parallel High Throughput DNA Sequencing: Automation for Microbial Community, Gene Expression and de novo Deciphering of New Genomes
Bruce A. Roe, Ph.D.,
George Lynn Cross Research Professor of Chemistry and Biochemistry, Advanced Center for Genome Technology, Stephenson Research
and Technology Center, University of Oklahoma

2. A Brief History of Long Re
ad Automated DNA Sequencing Instruments: ABI and 454/Roche
454/Roche GS-FLX-XLR
1Gb/run
0.04
0.5
1.0
1.5
2.0 ~ 10 20 30 40
1994
1996
1998
2000
2002
2004
2006
Date of Introduction
# Million Bases/Run
2007
ABI 370/377 40Kb/run 50 75
100
2008
454/Roche GS-FLX
100Mb/run
30Mb/run
454-GS20
ABI Mb/run
ABI Kb/run

3. 454 GS-FLX 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 Picotiter Plate
Load Enzyme Beads
Load beads into PicoTiter Plate
Centrifugation
44 μm

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

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
25-250bp ends of a circularized DNA molecule
Amplicon
PCR product for SNP discovery
Roe Lab
Combined Paired-End and Shotgun approach
Best of both worlds

11. Our Combined Paired End & Shotgun DNA Preparation Protocol Overview
Shear to 2-4 Kbp fragments on the Hydroshear
Quantitate on Caliper AMS-90 or by RealTime PCR
DNA End Repair & Linker Ligation as in paired-end protocol
Cleave the Terminal Linkers with EcoR1
Ligate to Circularized the DNA
Shear to ~500 bp fragments in the Nebulizer but eliminate the enrichment step for fragments containing linker

12. Our Combined Paired End/Shotgun DNA Preparation Protocol Overview (cont)
Quantitate on Caliper AMS-90 or by RealTime PCR
DNA End Repair, Adaptor Ligation,
Adapter End Repair
Amplification (emPCR)
Pyrosequencing of the combined linker-containing (paired end) and shotgun fragments on 454/Roche GS-FLX

13. Assembly of Sequence Reads from Our Combined Paired-End/Shotgun Protocol
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
~15% of the total reads contain the middle linker sequence
Assembly of the reads by Newbler
Convert paired ends for ordering and orienting
*.454f and *.454r

14. 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

15. 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):

16. DNA Purification through the Qiagen Minelute Columns vs
DNA Purification through the Qiagen Minelute Columns vs... Agencourt SPRI Magnetic Beads
Qiagen Minelute centrifuge column
Agencourt SPRI magnetic beads
At least a 30% increase in yield with the SPRI beads and it is easier to automate when using the SPRI beads

17. Homemade 96 well Magnetic Plate for Purification of the SPRI Beads
Inverted 96 well DNA sequencing plate with cylindrical magnets

18. Enzyme Chilling Station
Plastic rack fitted with Swagelock fittings and tubing for cooling.

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

20. Automated Library Making on the Caliper-Zymark SciClone
To view this automation, get our quicktime movie 454ZymarkPrep.mov

21. We also have increased the average read lengths from 250 to > 315 bases by increasing the number of flows and amounts of reagents
Slightly dilute the Substrate, Inhibitor and Apyrase by transferring 2.5mL from one of the Buffer CB bottles to each respective tube in the reagent tube-tray
Add 174ul (as opposed to 164ul) from the tube of apyrase to the apyrase buffer tube in the reagent tube-tray.
Transfer 150ml Buffer CB from bottle 3 (at the back of the cassette) to bottle 0 (at the front of the cassette).
Modify the run script to allow for 130 flow cycles

22. Reuse the Pico Titer plate after cleaning by sonication

23. Summary - Methods For library preparation, It is possible to:
incorporate both shotgun and paired end reads in the same library
replace the Qiagen Minelute centrifuge columns with Agencourt SPRI beads in the library preparation and build (or buy) an enzyme chilling station to facilitate automating the library making process
eliminate the steps involved in single stranded DNA preparation steps
It also is possible to:
break the emulsion after emPCR using centrifugation rather than using a Swinlock filter containing a sieving fabric.
Increase the volumes of the FLX reagents and increase the number of cycles results in a significantly increased read length.
reuse the PicoTiter plate after cleaning by sonication
All our protocols are available on our lab protocol web site at url:

24. Applications Whole Genome Sequencing Pooled samples EST Libraries
Plant viruses
Plant fungi
BAC-based genomic sequencing
EST Libraries
Bacterial Communities

25. Novel cDNA pooling strategy
Add tags to the PCR primer sequences to allow for deconvolution of viral sequences post sequencing
cDNA samples are pooled in sets with 24 unique individual tags after a two step PCR

26. 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 second Taq Polymerase PCR with one of the 24 four base 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

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

28. 10 Day Contour Clamped Homogenous Electrophoretic Field (CHEF) Gels for Chromosome Isolation
S.pombe
Po OkAlf-8 in all 4 lanes
Excise individual chromosomal bands, freeze at -200C and then melt by heating to 65 0C.
Mix 500 ul aliquots of TE saturated phenol and melted gel and re-freeze at -200C
Centrifuge at 2500 RPM in a table top centrifuge at -200C
Remove aqueous layer and extract any residual phenol twice with water-saturated ether
Ppt with 2.5 vol of 95% ethanol/acetate, wash 70% ethanol and dry the DNA
Dissolve the DNA in 10 ul of 10:0.1 TE
Chr. # 1 2
5.7 Mb 3 4
4.6 Mb 5 6 7
3.5 Mb

29. Eluted & amplified chromosomes on a 1% agarose gel
10 Day Contour Clamped Homogenous Electrophoretic Field (CHEF) Gels for Chromosome Isolation
Qiagen REPLI-g Mini kit was used to amplify the chromosomes
2.5 ul of the purified chromosomal DNA was mixed with 2.5 ul of Qiagen denaturation buffer for 3 minutes at 250C followed by mixing with 5 ul of Qiagen neutralization buffer.
A master mix containing 10 ul nuclease-free water, 29 ul reaction buffer (containing dNTPs and exonuclease-resistant primers) and 1 ul of the Qiagen’s DNA polymerase was added to the treated chromosomal DNA and incubated at 300C overnight.
The amplified chromosomal DNA product then was verified on a 1% agarose gel by electrophoresis and subjected to the mixed shotgun paired-end sequencing where over 90% of the sequences matched in our CRR database
Eluted & amplified chromosomes on a 1% agarose gel
BAC Hind Hind3

30. Summary of our use of CHEF gels for chromosome isolation and subsequent amplification for sequencing
Using our long established freeze/thaw phenol extraction protocol, individual chromosomes can be purified from chromosome grade agarose CHEF gels and then
Amplified using the Qiagen REPLI-g Mini kit
Sequence data can obtained after library making, emPCR and massively parallel pyrosequencing on the 454/Roche GS-FLX with over 90% of the sequences matching our target genome/fungal database

31. Strategy of adding the 454/Roche MID-based Tags prior to BAC Pooling
BAC growth in 96 deep well microtiter plates
Robotic BAC isolation via the cleared lysate protocol using the Hydra robot.
Sheer each BAC individually and create the paired end libraries on the Zymark SciClone robot.
Individually tagged A linkers are added with B linkers prior to pooling 12 tagged libraries, followed by
emPCR, and half-plate sequencing of each pool. 31

32. Strategy of adding the 454/Roche MID-based Tags prior to BAC Pooling
12 uniquely tagged individual shotgun libraries would be pooled and sequenced on each half- 454/Roche GS-FLX picotiter plate, 24 tagged libraries/full plate
Kb BACs requires 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 and no ABI 3730 runs are needed to deconvolute the individual BACs as each BAC is individually tagged
The BACs then are easily closed and finished using PCR-based methods. 32

33. Analysis of ordered and oriented combined shotgun/paired-end results
vector
repeat sequences missing in the 454 data but present in the 3730 and/or obtained by PCR-based closure
454/Roche GS-FLX only assembled sequences
Phrap-assembled ABI-3730 and 454/Roche GS-FLX sequences
Un-joined 454 data often with no missing base but joined by 454 paired-ends and spanned by 3730 or PCR-based sequences
Our present strategy is to use the combined shotgun/paired-end pyrosequencing approach on the 454/Roche GS-FLX followed by PCR-based closure methods. 33

34. Acknowledgments Collaborators
Plant Virus studies
Oklahoma State University: Ulrich Melcher, Vijay Muthamukar
Noble Foundation: Marilyn Roossinck, Guoan Shen, Byoung Min, Rick Nelson, Tracy Feldman
Phymatotrichopsis omnivora aka Cotton Root Rot Fungi
Oklahoma State University: Steve Marek
Noble Foundation: Carolyn Young
Medicago truncatula
University of Minnesota: Nevin Young, Roxanne Denny, Steven Cannon (now at Iowa State), Arvind Bhari, Shelly Wang
The JCV Institute: Chris Town, Foo Cheung
The John Innes Institute, UK: Giles Oldroyd & Sanger Institute: Jane Rogers
Toulouse/INRA & Genoscope, France: Fredric Debelle, Francis Quetier
Munich Bioinformatics Center IMGAG Consortium: Claus Mayer
Funding from the NSF Plant Genome, Microbial and EPSCoR Programs and the USDA

35. OU Genome Center Personnel
Automation
Graham
Fares
Doug
Simone
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