Data on file.For Research Use Only. Not for use in diagnostic procedures.

Sample Dilution

Prepare and Dispense Master Mixes

Reagent Preparation

Data Analysis

Add Samples and Controls

Perform qPCR

ACCURATE & REPRODUCIBLE

Benefits • 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 accurate, equimolar pooling for multiplexed sequencing

• Flexibility to support manual and automated, high-throughput pipelines; as well as PCR-free workflows

Accurate and reproducible quantification of NGS libraries prior to pooling and sequencing is critical for optimal and cost-effective use of sequencing capacity. KAPA Library Quantification Kits provide a proven, qPCR-based solution for the quantification of libraries prepared for sequencing on Illumina® and Ion Torrent™ platforms.

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.

KAPA Library Quantification KitsNext-generation qPCR meets next-generation sequencing

2Data on file.For Research Use Only. Not for use in diagnostic procedures.

The complete library quantification solution • Kits contain all the reagents needed for absolute, qPCR-based quantification of individual NGS libraries

or indexed library pools.

• Standard curves are designed to support all library construction workflows, including PCR-free methods.

• Only/all sequencing-competent library fragments are quantified, irrespective of over-amplification— whereas other methods under- or over-estimate library concentrations.

• Data analysis templates and assistance are available from sequencing.roche.com/support.

The principle of the KAPA Library Quantification Kit. Six pre-diluted DNA Standards and appropriately diluted NGS libraries are amplified using platform-specific qPCR primers that target adapter sequences. The average Cq value for each DNA Standard is plotted against its known concentration to generate a standard curve. The standard curve is used to convert the average Cq values for diluted libraries to concentration, from which the working concentration of each library is calculated.

qPCR quantifies all sequenceable molecules. (A) The ligation of adapters to insert DNA results in a mixed population of molecules, some of which do not support cluster amplification and sequencing. qPCR measures only those molecules with the correct adapter configuration, which is especially critical in PCR-free workflows. Spectrophotometric, fluorometric, and electrophoretic methods, measure all double-stranded molecules, irrespective of adapter configuration. (B) Excessive library amplification leads to primer depletion. Subsequent rounds of denaturation and annealing produces imperfectly annealed, partially double-stranded DNA heteroduplexes. Electrophoretic and fluorometric assays that employ dsDNA-binding dyes are designed to detect double-stranded DNA fragments only, leading to under-quantification of over-amplified libraries.

These unsequenceable molecules are detected by

Nanodrop,™ Bioanalyzer,® PicoGreen,® and Qubit®

assays which leads to overquantification

and underclustering.

Bioanalyzer, PicoGreen, and Qubit assays do not accurately detect these highly single-stranded sequenceable molecules, which leads to underquantification and overclustering.

1.7

1.5

1.3

1.1

0.9

0.7

0.5

0.3

0.1

-0.12 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Cycle

Nor

mal

ized

Flu

ores

cenc

e

Library sample

Std 1 Std 2 Std 3 Std 4 Std 5 Std 6

25

20

15

10

5

0

-4 -2 0 2

Log (concentration)

y = -3.4496x + 36.24R2 = 0.9998

PCR-free workflows

A B

Products of low libraryconstruction efficiency

Products of primer depletion

Workflows withlibrary amplification

3Data on file.For Research Use Only. Not for use in diagnostic procedures.

The engineered KAPA SYBR® FAST DNA Polymerase is the key to efficient library quantification • KAPA SYBR FAST was engineered to amplify diverse DNA fragments with similar efficiency.

• Library quantification kits containing wild-type DNA polymerases only count "easy" library molecules.

A proven track record for product quality and consistency • The properties of KAPA SYBR FAST enable the use of a single DNA Standard for all library types.

• Pre-diluted standards are produced with very high lot-to-lot consistency.

High consistency across different library types and across hundreds of production lots. (A) Nine human WGS libraries (41% GC) and two microbial WGS libraries (Rhodococcus sp., 70% GC and Staphylococcus sp., 35% GC) were quantified with distinct lots (Lots 1 and 2), and distinct sets of reagents from the same lot (Set 1 and 2) of KAPA Library Quantification Kit for the Illumina® platform. (B) Amplification efficiencies for 55 consecutive lots of KAPA DNA Standards released since September 2009.

KAPA SYBR FAST is engineered for high-efficiency library quantification. (A) Amplification efficiencies achieved with the KAPA SYBR FAST qPCR Mix for ten diverse amplicons, plotted against GC content. All ten amplicons were amplified with similar efficiency (within the optimal range of 95% – 105%). This confirms that the KAPA SYBR FAST enzyme is ideal for the amplification of heterogenous populations of DNA fragments, such as NGS libraries. (B) DNA fragment size distributions for an NGS library before (grey fill) and after qPCR amplification with the KAPA SYBR FAST qPCR Kit and two competitor kits containing wild-type Taq polymerase. Reactions were performed with the following cycling protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 45 sec. The KAPA SYBR FAST quantification product is representative of the template DNA (NGS library), whereas the quantification products generated with wild-type enzymes are not.

Input Amount

Rea

ctio

n ef

ficie

ncy

(%)

Reaction efficiency vs. GC-content

GC content (%)

40 45 50 55 60 6565

75

85

95

105

115

125

Fluo

resc

ence

0.025 0.20 0.5 1.00 2.00

Size distribution before qPCRKAPA SYBR FASTCompetitor 1Competitor 2

Size (kb)

30

25

20

15

10

5

0566 601 557 606 270 400 375 438 438 375 405

Con

cent

ratio

n (p

M)

Average fragment size (bp)

Human

Rhod

ococ

cus s

p.(~

70%

GC)

Stap

hylo

cocc

us sp

.(~

35%

GC)

Lot 1, set-1Lot 1, set-2Lot 2

Stan

dard

cur

ve e

ffici

ency

Lot s

ize

(num

ber

of s

tand

ard

curv

es)

Lot

0

20

40

60

80

100

0

50k

100k

150k

200k

250k

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55

A

A

B

B

Published by:

Roche Sequencing Solutions, Inc.4300 Hacienda DrivePleasanton, CA 94588

sequencing.roche.com

Data on file.For Research Use Only. Not for use in diagnostic procedures.KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners. ©2017 Roche Sequencing Solutions, Inc. All rights reserved. SEQ100005 A038 02/17

Reproducible cluster density and improved pooling for multiplexed sequencing from samples of variable quality• Improved library quantification enables optimal and more predictable cluster densities, to maximize

sequencing capacity and throughput.• Accurate and sensitive quantification facilitates equimolar pooling of indexed libraries, irrespective

of pre-pooling library concentrations.• Uniform distribution of reads across all the libraries in a pool optimizes sequencing costs.• Reliable results are achievable for all library types, including those prepared from low-quality FFPE DNA.

Accurate and reproducible library quantification enables improved and consistent cluster densities. The implementation of KAPA Library Quantification Kit into the Illumina® GA sequencing workflow at The Broad Institute (Cambridge, MA, USA) significantly reduced cluster density variability and eliminated the need for titrations. Average number of clusters per tile are shown for consecutive libraries.

Accurate and sensitive quantification enables a uniform distribution of reads in muliplexed sequencing pools. Twenty-four indexed libraries were quantified by qPCR using the KAPA Library Quantification Kit (for Illumina® platforms) and combined to create three sequencing pools of equimolar concentration. Each pool was sequenced in a different lane of the same flow cell on a HiSeq® 2500 instrument. (A) The twenty-four individual libraries represented a ~44-fold range of pre-pooling concentrations (5.2 nM to 229.8 nM), whereas the sequencing read distribution (B) only varied between 9.6% and 13.9%. The coefficient of variation (CV) for pre-pooling library concentration was 94.9%, 49.4%, and 127.9% for the libraries pooled for sequencing in lanes 1, 2, and 3, respectively. The KAPA Library Quantification Kit enabled accurate equimolar pooling, reducing the CV to 2.5%, 4.8%, and 11.2% after normalization.

Conv

ersio

n (%

)

600

500

400

300

200

100

020 40 60 800

Raw

clu

ster

s [1

,000

k/m

m2 ]

Implementation of KAPA Library Quantification Kit

Sample number

nM

Final library concentration

50

100

150

200

250

0

83.6 ± 79.4

16.3 ± 8.013.8 ± 17.6

2 3 4 5 6 71 8 10 11 12 13 14 159 16 18 19 20 21 22 2317 24

Lane 1 Lane 2 Lane 3

02468

10121416

By

inde

x, %

Total assigned reads in lane

12.5 ± 0.3 12.5 ± 0.6 12.5 ± 1.4

2 3 4 5 6 71 8 10 11 12 13 14 159 16 18 19 20 21 22 2317 24

Lane 1 Lane 2 Lane 3

Ordering information

Roche Cat. No. KAPA Code Description qPCR Instrument/Reference Dye

07960140001 KK4824 KAPA Library Quantification Kit - Illumina Universal

07960204001 KK4835 KAPA Library Quantification Kit - Illumina ABI Prism®

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

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

07960336001 KK4873 KAPA Library Quantification Kit - Illumina ROX Low

07960158001 KK4827 KAPA Library Quantification Kit - Ion Torrent PGM Universal

07960212001 KK4838 KAPA Library Quantification Kit - Ion Torrent PGM ABI Prism®

07960263001 KK4847 KAPA Library Quantification Kit - Ion Torrent PGM Bio-Rad iCycler™

07960301001 KK4857 KAPA Library Quantification Kit - Ion Torrent PGM Roche LightCycler® 480

07960344001 KK4874 KAPA Library Quantification Kit - Ion Torrent PGM ROX Low

*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.

A

B