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

3

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

12

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

20

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.