introducing bigdye terminator v3.1 and v1.1 kits

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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

4

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)

5

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

8

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

9

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

10

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

11

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

13

Long Read Length with BigDye® Terminator v3.1 Chemistry on the 3730xl DNA Analyzer, 50 cm array, and POP-7™ Polymer

14

Customer Example of Improved Performance With BigDye® Terminator v1.1 Chemistry–Reading Through Region With High G/T

Kits

v1.0

v1.1

15

Customer Example of Improved Performance With BigDye® Terminator v1.1 Chemistry–Reading Through C/T Repeat

Kits

v1.0

v1.1

16

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

17

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

18

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

19

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

21

Complete Removal of Unincorporated Terminators From 1X v3.1Chemistry Reactions With Ethanol/EDTA Precipitation Protocol

V3.0

V3.1

22

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

24

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

25

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.

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