Next-generation DNA Sequencing meets Next-generation qPCR

KAPA LIBRARY QUANTIFICATION KITS

KAPA Library Quantification Kits contain all the reagents needed for NGS library amplification using absolute, qPCR-based quantification. This includes KAPA SYBR® FAST qPCR Master Mix (formulated with different passive reference dyes for different qPCR instruments), a platform-specific library quantification primer premix, and a pre-diluted set of DNA standards.

• reliable and sensitive quantification of all sequencing-

competent library molecules

• accurate and reproducible quantitation across a wide

range of library types, concentrations, fragment length

distributions and GC content

• more efficient, equimolar pooling for multiplexed sequencing

• flexibility to support manual and automated, high-

throughput pipelines; as well as PCR-free workflows

Benefits include:

Unless otherwise stated in references, presented data on file For Research Use Only. Not for use in diagnostic procedures.

KAPA Library Quantification Kits | 2For Research Use Only. Not for use in diagnostic procedures.

Unless otherwise stated in references, presented data on file

1 hr 00 min

(Automated liquid handling platform)

1 hr 30 min

30 min

Total time: 3 hr 00 min

Starting material

Sample fragmentation

Library prep

Library QC (NanoDrop/Agilent)

Titration flow cell

Titration cluster amplification

Bulk flow cell

Bulk cluster amplification

Initiate sequencing

Standard Illumina GA workflow HTP sample workflow at the Broad Institute (qPCR library quatiffication)

KAPA Library Quantification Kit

Library DNA dilutions

Load qPCR plate

run qPCR protocol

Analyze data

Day 1

Day 2

Day 3

Day 4

qPCR Library Quantification Results in Streamlined WorkflowsKAPA Library Quantification Kits eliminate the need for time-consuming and expensive titrations and provide a conducive format for streamlining high-throughput workflows.

Standard Illumina GA workflow Streamlined workflow with qPCR library quantification

Current standard protocols for commercial next generation sequencing platforms employ laborious, costly, and unreliable methods for quantifying DNA libraries.Accurate quantification of PCR-competent sequencing templates is crucial for reliable clonal amplification via either emulsion

PCR (emPCR) or bridge PCR (bPCR) - underestimation results in non-clonality, while overestimation leads to inefficiency via poor

yields of clonally amplified templates.

Standard methods for quantifying NGS libraries have a number of important disadvantages. Electrophoresis and spectrophotometry measure total nucleic acid concentrations, whereas optimal cluster density or template-to-bead ratio depend on the appropriate concentration of PCR-amplifiable DNA molecules. These methods also have low sensitivity, consuming nanograms of precious samples, and are not suitable for high-throughput workflows.

Quantitative PCR (qPCR) is inherently well-suited for next-generation sequencing library quantification:

• qPCR specifically quantifies only PCR-competent DNA molecules,

• is highly sensitive allowing accurate quantification of low concentration libraries,

• is amenable to automated liquid handling.

KAPA Library Quantification Kits are optimized for the Illumina® Genome Analyzer, Roche 454 Titanium series, and Roche 454

FLX series platforms and include defined, reliable DNA concentration standards and state-of-the-art qPCR reagents, containing

a DNA polymerase engineered for SYBR® Green-based qPCR through a process of molecular evolution.

KAPA Library Quantification Kits | 3For Research Use Only. Not for use in diagnostic procedures.

Unless otherwise stated in references, presented data on file

Reliable Quantification Results in Consistent Cluster Density

"Before qPCR was adopted for library quantification, cluster density was extremely variable. Implementation of the KAPA Library Quantification Kit in our sequencing workflows resulted in a significant reduction in variability across multiple libraries, negating the need for cluster amplification titration runs."

- Broad Institute, Cambridge, MA U.S.A.

Cluster density before and after implementation of the KAPA Library Quantification Kit (right). The implementation of KAPA Library Quantification Kits into the Illumina® GA sequencing workflow at the Broad Institute significantly reduced cluster density variability and eliminated the need for titrations. Average number of clusters per tile are shown for consecutive ibraries. Data courtesy of the Broad Institute

Efficient Amplification of a Wide Range of Templates during qPCRTraditional qPCR reagents are optimized for short amplification targets; longer targets, unbalanced GC-content, and problematic secondary structures may result in low amplification efficiency and unreliable quantification of some library molecules. To address the demands of quantifying complex DNA libraries, Kapa Biosystems has engineered a DNA polymerase specifically for SYBR® Green-based qPCR, enabling efficient amplification of targets that present a challenge to wild-type enzymes. KAPA Library Quantification Kits contain this engineered polymerase to ensure robust amplification of longer fragments, across a broad range of GC-content, required for accurate library quantification.

Fragment size distributions before and after qPCR. Fragment size distributions before (grey fill) and after qPCR amplification using three commercial qPCR master mixes (KAPA SYBR® FAST (green), Competitor F (orange), and Competitor G (pink)). Competitor kits contained wild-type Taq polymerase. Reactions were performed with the following cycling protocol: 95ºC for 10 min followed by 40 cycles of 95ºC for 10 sec and 60ºC for 45 sec.

Robust amplification translates into accurate qPCR quantification of diverse libraries. The KAPA Libary Quantificaiton Kit was used to determine the concentration of two Illumina GA libraries with unusual GC content (Rhodococcus sp.; ~70% GC – shown above, Staphylococcus sp.; ~35% GC – not shown). Both libraries amplified with efficiency >95%. Two-fold dilution series (1:1000 through 1:16000) were prepared in triplicate, and qPCR performed according to the recommendations in the product technical data sheet.

Fluo

resc

ence

Size (kb)0.025 0.20 0.5 1.00 2.00

Slow distribu�on before qPCRKAPA SYBR FASTCompe�tor-FCompe�tor-G

12.00

11.00

10.00

8.00

7.00

9.00

1:16000 1:8000 1:4000 1:2000 1:10006.00

R2: 0.988

Efficiency: 97%

Rhodococcus sp. (~70% GC)

Ct

Dilution

Reliable DNA Quantification Standards with Minimal Variability From Lot-to-lot

Lot-to-lot variability of the KAPA Library Quantification Kit for the Roche Titanium series platform. Three distinct lots (orange, pink, blue) were compared by analyzing amplification plots of each set of quantification standards. Triplicates of each data point were averaged.

Nor

m. F

luor

o.

Cycle

10-0.0

10-0.1

10-0.2

10-0.4

10-0.5

10-0.7

10-0.3

5 10 15 20 25 30 35

10-0.6

10-0.8

10-0.9

R2: 0.999

Efficiency: 99%

Slope: -3.334

Minimal lot-to-lot and kit-to-kit variability. Nine human DNA libraries and two microbial DNA libraries were used to compare quantification results obtained with distinct lots ("Lot 1" and "Lot 2"), and distinct sets of reagents from the same lot ("set 1" and "set 2") of KAPA Library Quantification Kits for the Illumina GA platform.

Ordering Information

Roche Cat. No. Kapa Code Description* qPCR Instrument

07960140001 KK4824 KAPA Library Quantification Kit - Illumina GA Universal

07960204001 KK4835 KAPA Library Quantification Kit - Illumina GA ABI Prism®

07960255001 KK4844 KAPA Library Quantification Kit - Illumina GA Bio-Rad iCycler™

07960298001 KK4854 KAPA Library Quantification Kit - Illumina GA Roche LightCycler® 480

07960131001 KK4821 KAPA Library Quantification Kit - Roche 454 Titanium Universal

07960182001 KK4831 KAPA Library Quantification Kit - Roche 454 Titanium ABI Prism®

07960247001 KK4841 KAPA Library Quantification Kit - Roche 454 Titanium Bio-Rad iCycler™

07960280001 KK4851 KAPA Library Quantification Kit - Roche 454 Titanium Roche LightCycler® 480

07960123001 KK4820 KAPA Library Quantification Kit - Roche 454 FLX Universal

07960174001 KK4830 KAPA Library Quantification Kit - Roche 454 FLX ABI Prism®

07960239001 KK4840 KAPA Library Quantification Kit - Roche 454 FLX Bio-Rad iCycler™

07960271001 KK4850 KAPA Library Quantification Kit - Roche 454 FLX Roche LightCycler® 480

*All kits contain 5 mL KAPA SYBR® FAST qPCR Master Mix (2X), 1 mL Primer Premix, and 6 x 80 uL DNA Quantification Standards. Kits contain primers, DNA standards, and qPCR reagents specific for both DNA sequencing platform and qPCR instrument. Primer Premix and DNA Quantification Standards are also sold separately.

30

25

20

15

10

5

0 566 601 557 606 270 400 375 438 438 375 405

Co

nce

ntr

atio

n (p

M)

Average fragment size (bp)

Human

Rho

doco

ccus

sp.

(~7

0%

GC

)

Stap

hylo

cocc

us sp

.(~

35

% G

C)

Lot 1, set-1

Lot 1, set-2

Lot 2

Unless otherwise stated in references, presented data on file© 2016 Kapa Biosystems. All trademarks are the property of their respective owners. SS104001 A179 4/16 For Research Use Only. Not for use in diagnostic procedures.

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