introducing bigdye terminator v3.1 and v1.1 kits
TRANSCRIPT
Introducing BigDye®
Terminator v3.1 and v1.1 kits
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BigDye® v3.1 and v1.1 Kit Improvements over v3.0 and “v1.0” Kits
- Greater success with difficult templates
- Longer, higher quality reads
- Enhanced Robustness
- Greater Peak height uniformity
- Optimized Signal Balance
Improvement Result
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BigDye® v3.1 and v1.1 Cycle Sequencing Kit Characteristics
- v3.1 and v1.1chemistries are reformulations of v3.0 and “v1.0” chemistries
- No new software or instrument recalibration required for use*
- Optimized protocols will enhance performance
- Sequencing buffer formulated specifically for v3.1 and v1.1 chemistries
*Note: Assumes instrument currently has corresponding BigDye ® chemistry calibration files
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BigDye® v3.1 and v1.1 Kits: Solutions for today’s wide range of sequencing applications
BigDye® v3.1 kitApplication V3.1 V1.1
de novo Sequencing + !
Resequencing + !
Sequencing difficult templates + +
Long-read sequencing + ! Sequencing across all template types (plasmids, BACs, and fosmids)
+ !
Mixed base detection +* ! * Sequencing short PCR products using rapid electrophoresis run modules
! +
+ Recommended ! Satisfactory * For this application customers may find that both chemistries will meet their needs
- for Most Applications- v3.0 kit v3.1 kit
- for Specialty Applications- “v1.0” kit v1.1 kit
BigDye® v1.1 kit
- Note: v2.0 kit customers should select new chemistry by application(s)
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Customer 1: Data Shows Greater Number of Phred Q20 Bases with v3.1 Chemistry
Phred Q20 Bases (Cust 1)
0100
200300
400500600
700800
9001000
Sample
Nu
mb
er o
f P
hre
d Q
20 B
ases
v3.0 v3.1
Chemistry Average Q20v3.0 629v3.1 840
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Customer Example of More Q20 Bases With a Difficult Template Using BigDye®
Terminator v3.1 ChemistryKits
v3.0
v3.1
Data courtesy of Dr. Kevin McKernan, Agencourt
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Customer Example of Increased Q Values Leading to More Q20 Bases
0
10
20
30
40
50
60
1 101 201 301 401 501 601 701 801 901
base
qu
alit
y n
um
ber v3.1
v3.050 per. Mov. Avg. (v3.1)50 per. Mov. Avg. (v3.0)
Data Courtesy of Dr. Tim Holzer,The Whitehead Institute/MIT Center for Genome Research
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Customer 2: Data Shows Greater Number of Phred Q20 Bases with v3.1 Chemistry
Phred Q20 Bases (Cust 2)
0
100
200
300
400
500
600
700
800
900
1000
1 Sample
Num
ber o
f Phr
ed Q
20 B
ases
v3.0 v3.1
Data Courtesy of Dr. Elaine Mardis, Washington Univerisity Chemistry Average Q20
v3.0 526v3.1 716
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Customer Example of More Q20 Bases With a Difficult Template Using BigDye®
Terminator v3.1 Chemistry
Kits
v3.0
v3.1
Data courtesy of Dr. Elaine Mardis, Washington University
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Customer 3: Data Shows Greater Number of Phred Q20 Bases with v3.1 Chemistry
Phred Q20 Bases (Cust 3)
0
100
200
300
400
500
600
700
800
900
Sample
Num
ber o
f Phr
ed Q
20 B
ases
v3.0 v3.1
Chemistry Average Q20v3.0 393v3.1 677
Data Courtesy of Dr. Donna Muzny, Baylor College of Medicine
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Customer example of BigDye® Terminator v3.1 Chemistry Reading Through G-rich Region
Kits
v3.0
v3.1
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Long Read Length with BigDye® Terminator v3.1 Chemistry on the 3100 Genetic Analyzer, 80 cm array, and POP-4™ Polymer
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Long Read Length with BigDye® Terminator v3.1 Chemistry on the 3730xl DNA Analyzer, 50 cm array, and POP-7™ Polymer
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Customer Example of Improved Performance With BigDye® Terminator v1.1 Chemistry–Reading Through Region With High G/T
Kits
v1.0
v1.1
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Customer Example of Improved Performance With BigDye® Terminator v1.1 Chemistry–Reading Through C/T Repeat
Kits
v1.0
v1.1
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Customer Example of More Q20 Bases With Difficult Templates Using BigDye®
Terminator v3.1 ChemistryKits
v3.0
v3.1
Data courtesy of Dr. Elaine Mardis, Washington University
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Improved Peak Height Uniformity With Customer Samples
Data courtesy of DNA Sequencing Core Lab, University of Utah*Note: 100% peak height uniformity represents idealized situation in which all analyzed data peaks are of equivalentheight
Peak Height Uniformity
50%
55%
60%
65%
70%
75%
80%
85%
Sample
Per
cent
Uni
form
ity
v3.0 v3.1
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Core Lab Customer Example of BigDye®
Terminator v3.1 Chemistry Sequencing a Difficult Template
Kits
v3.0
v3.1
Data courtesy of DNA Sequencing Core Lab, University of Utah
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Mixed Base Calling with v3.1 kit –MicroSeq® Software Template
Kits
v3.0
v3.1
Samples run on 3700 instrument with POP-6™ Polymer
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Customer Sample Showing Mixed Base Sequence Detection with v1.1 Chemistry
V1.0
V1.1
Data Courtesy of Dr. Suzanne Cordovado, CDC. Samples were run on a 3100 with a 36 cm array with POP™-6
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Complete Removal of Unincorporated Terminators From 1X v3.1Chemistry Reactions With Ethanol/EDTA Precipitation Protocol
V3.0
V3.1
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Complete Removal of Unincorporated Terminators From 1X v3.1 Chemistry Reactions WithSpin Column/SDS Protocol
V3.0
V3.1
Edge Biosystems, 96 well DTR Plate
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Complete Removal of Unincorporated Terminators From 1X v3.1 ChemistryReactions Using CleanSeq™ Reagent
V3.0
V3.1
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Conclusions: BigDye® Terminator v3.1 and v1.1 Kits
- Chemistries offer improved robustness, optimized signal balance, and greater peak height uniformity
- Improvements will result in longer reads and higher success rates
- Kits will enable experiments to be completed in less time with fewer runs, thus lowering the cost of sequencing
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Acknowledgements
R&D•Diane Bond
•Cheryl Heiner
•Kerrie Lee
•Allen Swei
•Wei-Hsien Yang
CDM•Jon Cassel
•Jaime Lee
•Yenyoung Lei
•Kitland Louie
•Kathy Perry
•John Teare
Marketing•Eric Ferguson
Statistics•Chris Schwarz
Service•Mark Garrett
Specialists•Mike Hughes
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- For Research Use Only. Not for use in diagnostic procedures.
- ABI PRISM, Applied Biosystems, BigDye and MicroSeq are registered trademarks and AB(Design), Applera, POP-4, and POP-7 are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.