1 xt Gen. Sequencing Sept. 24, 2008 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 Researchand Technology Center, University of Oklahoma
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Next Gen. Sequencing Sept. 24, 2008 1 Massively Parallel High Throughput DNA Sequencing: Automation for Microbial Community, Gene Expression and de novo.
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1Next Gen. Sequencing Sept. 24, 2008
Massively Parallel High Throughput DNA Sequencing: Automation for
Microbial Community, Gene Expression and de novo Deciphering
of New Genomes
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 Researchand Technology Center, University of Oklahoma
2Next Gen. Sequencing Sept. 24, 2008
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
A Brief History of Long Read Automated DNA Sequencing
Instruments: ABI and 454/Roche
A Brief History of Long Read Automated DNA Sequencing
DNA Purification through the Qiagen Minelute Columns vs... Agencourt SPRI Magnetic BeadsDNA 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
17Next Gen. Sequencing Sept. 24, 2008
Homemade 96 well Magnetic Plate for Purification of the SPRI Beads
Homemade 96 well Magnetic Plate for Purification of the SPRI Beads
Inverted 96 well DNA sequencing plate with cylindrical magnets
18Next Gen. Sequencing Sept. 24, 2008
Enzyme Chilling StationEnzyme Chilling Station
Plastic rack fitted with Swagelock fittings and tubing for cooling.
19Next Gen. Sequencing Sept. 24, 2008
Zymark SciClone Deck Arrangement
Shaker
EtOH
Enzyme Mixes
Shaker
Shaker
Magnet
SPRI Beads
Sample
Buffers
Waste
20Next Gen. Sequencing Sept. 24, 2008
QuickTime™ and a decompressor
are needed to see this picture.
Automated Library Making on the Caliper-Zymark SciCloneAutomated Library Making on the Caliper-Zymark SciClone
To view this automation, get our quicktime movie 454ZymarkPrep.mov
21Next Gen. Sequencing Sept. 24, 2008
We also have increased the average read lengths from 250 to > 315 bases by increasing the number of flows and amounts of reagents
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
22Next Gen. Sequencing Sept. 24, 2008
Reuse the Pico Titer plate after cleaning by sonication
Reuse the Pico Titer plate after cleaning by sonication
23Next Gen. Sequencing Sept. 24, 2008
Summary - Methods 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: http://www.genome.ou.edu/proto.html
• 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
26Next Gen. Sequencing Sept. 24, 2008
Strategy for preparing cDNA ready for 454 sequencing from dsRNA
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’ 3’
5’3’NNNNNN
CCTTCGGATCCTCC
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
3’ 5’
5’
GGAAGCCTAGGAGG
5’
5’
CCTCCTAGGCTTCCGAGA
+5’
3’ 5’CCTCCTAGGCTTCCNNNNNN
CCTCCTAGGCTTCC
NNNNNN
NNNNNNCCTTCGGATCCTCC5’ 3’
+
Additional Rounds of RT PCR with Random Hexamer Primers
NNNNNN
CCTTCGGATCCTCC
CCTCCTAGGCTTCC
NNNNNN
CCTCCTAGGCTTCCNNNNNN
NNNNNNCCTTCGGATCCTCC5’ 3’
AGAGCCTTCGGATCCTCC
GGAAGCCTAGGAGG
+ 5’ 3’
3’ 5’
AGAGCCTTCGGATCCTCC
CCTCCTAGGCTTCCGAGA
Amplified Product Ready for Ligating 454 A and B Primers
A B
27Next Gen. Sequencing Sept. 24, 2008
Uniquely Tagged cDNA Sample on the 454
Uniquely Tagged cDNA Sample on the 454
454 tag (TCAG)
TGP Unique tag (GACA)
TGP common primer
(CCTTCGGATCCTCC)
RT-PCR Sequence
28Next Gen. Sequencing Sept. 24, 2008
10 Day Contour Clamped Homogenous Electrophoretic 10 Day Contour Clamped Homogenous Electrophoretic Field (CHEF) Gels for Chromosome IsolationField (CHEF) Gels for Chromosome Isolation
3.5 Mb
4.6 Mb
5.7 Mb
12
3
567
4
S.pombe Po OkAlf-8 in all 4 lanes
Chr. #• 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
29Next Gen. Sequencing Sept. 24, 2008
Eluted & amplified chromosomes on a 1% agarose gel
BAC Hind3 1 2 3 4 5 6 7 Hind3
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
10 Day Contour Clamped Homogenous Electrophoretic 10 Day Contour Clamped Homogenous Electrophoretic Field (CHEF) Gels for Chromosome IsolationField (CHEF) Gels for Chromosome Isolation
30Next Gen. Sequencing Sept. 24, 2008
Summary of our use of CHEF gels for Summary of our use of CHEF gels for chromosome isolation and subsequent chromosome isolation and subsequent
amplification for sequencingamplification 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
31Next Gen. Sequencing Sept. 24, 2008
• 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.
Strategy of adding the 454/Roche MID-based Tags
prior to BAC Pooling
Strategy of adding the 454/Roche MID-based Tags
prior to BAC Pooling
32Next Gen. Sequencing Sept. 24, 2008
Strategy of adding the 454/Roche MID-based Tags
prior to BAC Pooling
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
• 24 150 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.
33Next Gen. Sequencing Sept. 24, 2008
Analysis of ordered and oriented combined shotgun/paired-end results
Analysis of ordered and oriented combined shotgun/paired-end results
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.
vector
454/Roche GS-FLX only assembled sequences
repeat sequences missing in the 454 data but present in the 3730 and/or obtained by PCR-based closure
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
34Next Gen. Sequencing Sept. 24, 2008
Acknowledgments• Collaborators
– Plant Virus studies• Oklahoma State University: Ulrich Melcher, Vijay Muthamukar• Noble Foundation: Marilyn Roossinck, Guoan Shen, Byoung Min, Rick Nelson,