Technical Data Sheet

KAPA Library Quantification Kit Kapa/Roche Kit Codes and Components

KK4824 – 07960140001
Illumina® Platforms Universal qPCR Master Mix

KR0405 – v7.16 ROX High (50X) and ROX Low (50X)

supplied separately
Complete Kits with:
This Technical Data Sheet provides product information DNA Standards KK4835 – 07960204001
and a detailed protocol for the KAPA Library Quantification 1 – 6 (80 µL each) ABI Prism™ qPCR Master Mix
Kits for Illumina platforms. Primer Mix (1 mL) KK4844 – 07960255001
KAPA SYBR FAST
® Bio-Rad iCycler™ qPCR Master Mix
Contents qPCR Master Mix KK4873 – 07960336001
(5 mL)
ROX Low qPCR Master Mix
Product Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
KK4854 – 07960298001
Product Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 qPCR Master Mix optimized for
LightCycler® 480
Product Compatibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
KK4923 – 07960441001
Product Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Universal qPCR Master Mix
Shipping and Storage . . . . . . . . . . . . . . . . . . . . . . . . . 2 ROX High (50X) and ROX Low (50X)
supplied separately
Handling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kits with: KK4933 – 07960484001
Quality Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Primer Mix (1 mL) ABI Prism qPCR Master Mix
Important Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . 3 KAPA SYBR FAST KK4943 – 07960522001
Accurate Liquid Handling. . . . . . . . . . . . . . . . . . . . . . . 3 qPCR Master Mix Bio-Rad iCycler qPCR Master Mix
(5 mL)
Sample Concentration and Dilutions. . . . . . . . . . . . . . 3 KK4973 – 07960727001
ROX Low qPCR Master Mix
Sample Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
KK4953 – 07960573001
Contamination and No-template Controls. . . . . . . . . . 3
qPCR Master Mix optimized for
Reaction Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 LightCycler 480
Internal Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Kits with: KK4903 – 07960409001
Replicates, Data Reliability, Throughput, DNA Standards (80 µL) DNA Standards 1 – 6
and Per-sample Cost. . . . . . . . . . . . . . . . . . . . . . . . . . 4 Kits with: KK4906 – 07960417001
Assay Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Dilution Control (80 µL) DNA Standard 0

Process Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Quick Notes

Detailed Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
• The DNA Standards provided in the kit represent a
Data Analysis and Interpretation. . . . . . . . . . . . . . . . . . . . 9 10-fold dilution series (20 pM to 0.0002 pM).
Restrictions and Liabilities. . . . . . . . . . . . . . . . . . . . . . . 12 • Ensure that the libraries to be quantified are
Note to Purchaser: Limited Product Warranty . . . . . . . . 12 compatible with the qPCR quantification primer
sequences given on the next page.
Note to Purchaser: Limited License . . . . . . . . . . . . . . . . 12
• Select the correct version of KAPA SYBR FAST qPCR
Master Mix for the qPCR instrument to be used.
• Accurate liquid handling is critical for reliable and
reproducible results.
• Refer to the KAPA Library Quantification Technical
Guide for a more in-depth discussion of the various
factors affecting accurate library quantification.

Effective date: October 2016 For Research Use Only. Not for use in diagnostic procedures.
KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Product Description recommended for high-throughput NGS pipelines. Please
refer to the KAPA Library Quantification Technical Guide
KAPA Library Quantification Kits for Illumina platforms for more information.
provide all the reagents needed for absolute, qPCR-based
quantification of Illumina libraries flanked by the P5 and
P7 flow cell oligo sequences. Kits contain:
Product Compatibility
KAPA Library Quantification Kits for Illumina platforms are
• Library Quantification DNA Standards 1 – 6 (a 10-fold
suitable for the quantification of any NGS library (prepared
dilution series of a linear, 452 bp template)
for Illumina sequencing) which contains the P5 and P7
• Library Quantification Primer Premix (10X), containing flow cell sequence motifs. Libraries constructed using
the following primers: full-length universal or indexed TruSeq™ adapters can
Primer 1: 5'-AAT GAT ACG GCG ACC ACC GA-3' be quantified after adapter ligation. Libraries prepared
with partial or stem-loop adapters can only be quantified
Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3'
with this kit after library amplification, during which
• KAPA SYBR® FAST qPCR Master Mix (2X), available indexes (and flow cell sequences) are added. To ensure
with various passive reference dyes (Table 1). compatibility, compare the adapter sequences used
Library quantification is performed by amplifying the set of during library preparation with the library quantification
six pre-diluted DNA Standards and diluted library samples primer sequences in Product Description.
by qPCR, using the KAPA SYBR FAST qPCR Master Mix Ensure that the correct KAPA SYBR FAST qPCR Master
and primers targeting the Illumina P5 and P7 flow cell oligo Mix is used, in accordance with the reference dye
sequences. The average Cq score for each DNA Standard requirements (if any) of the qPCR instrument to be used
is plotted against log10(concentration in pM) to generate for library quantification (Table 1).
a standard curve. The concentrations of diluted library
samples are then calculated against the standard curve, Product Specifications
using absolute quantification.
Shipping and Storage
KAPA Library Quantification Kits are rigorously tested to KAPA Library Quantification Kits are shipped on dry
ensure minimal lot-to-lot variation. The kit contains the ice or ice packs, depending on the destination country.
novel KAPA SYBR FAST DNA Polymerase, engineered Upon receipt, immediately store all components at
through a process of directed evolution for high- -15ºC to -25ºC in a constant temperature freezer. When
performance SYBR Green I-based qPCR. The ability stored under these conditions and handled correctly, the
of the engineered polymerase to amplify diverse DNA kit components will retain full activity until the expiry date
fragments with similar efficiency enables the use of a indicated on the kit label.
universal standard for the reliable quantification of all
Illumina libraries with an average fragment length of up to Handling
1 kb, irrespective of library type or GC content. Always ensure that components have been fully thawed
and thoroughly mixed before use. The KAPA SYBR FAST
KAPA SYBR FAST is an antibody-mediated hot start DNA
qPCR Master Mix may not freeze solidly, even when stored
polymerase formulation. KAPA Library Quantification Kits
at -15ºC to -25ºC.
are therefore suitable for use with automated liquid handling
systems for high-throughput sample quantification. The SYBR Green I dye (contained in the KAPA SYBR
FAST qPCR Master Mix) and ROX dyes are light sensitive.
Product Applications Exposure to direct light for an extended period of time will
result in loss of fluorescent signal intensity.
KAPA Library Quantification Kits for Illumina platforms are
designed for the accurate and reproducible quantification All components of KAPA Library Quantification Kits, as
of libraries prepared for Illumina sequencing. Any library well as the combined KAPA SYBR FAST/Primer Premix
with a concentration >0.0002 pM that contains sequences solution, are stable through 30 freeze-thaw cycles.
complementary to the primers in the Primer Premix (10X) Ensure that all reagents are stored protected from light
can be quantified with the kit, irrespective of the library at -15ºC to -25ºC when not in use. When protected from
type, how it was constructed, or on which Illumina light, reagents are stable in the dark at 2ºC to 8ºC for
instrument it will be sequenced. long-term storage, and may be stored at this temperature
for short-term use, provided that they do not become
The kit supports quantification of libraries across a range contaminated with microbes and/or nucleases.
of GC contents and average fragment lengths up to 1 kb.
Quality Control
In addition to NGS library quantification, the kit can also
All kit components are subjected to stringent functional
be used to detect library contamination in work spaces
quality control, are free of detectable contaminating exo-
used during the preparation of Illumina libraries.
and endonuclease activity, and meet strict requirements
The KAPA Library Quantification assay consists of with respect to DNA contamination. Please contact
repetitive pipetting steps, which can easily be automated. Technical Support at kapabiosystems.com/support for
The use of an automated liquid handling system is highly more information.

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

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Important Parameters it must be repeated with a more appropriate dilution of
the library. If multiple dilutions were included, those that
KAPA Library Quantification Kits enable accurate fall within the dynamic range of the assay can be used to
and reproducible qPCR-based quantification of quantify the library.
libraries prepared for Illumina sequencing. Successful
implementation of the assay in NGS workflows depends Library dilutions should be based on estimations from
on a number of factors, which are discussed below. For previous experience with libraries of the same type, or
more detailed information, please refer to the KAPA Library prepared using similar workflows, and/or on concentration
Quantification Technical Guide or contact Technical information obtained with other methods during library
Support at kapabiosystems.com/support. construction and quality control (e.g., those employing
NanoDrop™, Qubit® or Bioanalyzer). Refer to Comparison
Accurate Liquid Handling with Other Quantification Methods for more information.
Since qPCR is a very sensitive technique, and the dynamic
range of this assay extends to very low template copy Sample Quality
numbers, the reliability of results is highly dependent on Since dilute DNA degrades in an unbuffered environment,
accurate liquid handling. Care must be taken to ensure the libraries and controls must be stored and diluted in a
highest degree of accuracy when executing this protocol. buffered solution, such as 10 mM Tris-HCl, pH 8.0 – 8.5
This can be achieved as follows: (25°C). Tween® 20 (0.05%) may be included in the dilution
buffer to improve pipetting accuracy and reduce DNA
• Always ensure that reagents and samples are adsorption to plastic tubes and pipette tips. Never dilute
fully thawed and thoroughly mixed before use. libraries or controls with water.
After thawing and mixing, centrifuge tubes briefly to
remove any droplets from tube walls. Prepare fresh dilutions for each assay and keep dilutions on
ice during qPCR setup. Calculated library concentrations
• Concentrated solutions of DNA may be viscous, may be highly variable and/or inaccurate if diluted samples
making it difficult to accurately dispense small volumes are stored at room temperature or for long periods of time
for analysis. Avoid making extremely large dilutions (even at 4°C) prior to setting up qPCRs. If a sample has to
during sample preparation—see Detailed Protocol be re-assayed, fresh dilutions should be prepared for the
(step 2). If samples require very large dilutions to fall repeat assay.
within the dynamic range of the assay, it is preferable
to perform serial dilutions (e.g., make two consecutive Contamination and No-template Controls
1:100 dilutions instead of a single 1:10,000 dilution). Observe good laboratory practice at all times to avoid
• If possible, avoid the use of multi-channel pipettes. contamination of work areas, reagents, consumables, and
equipment with libraries, DNA Standards, or amplicons.
• Use a new pipette tip for each pipetting step, especially It is highly recommended that no-template controls
when dispensing the DNA Standards and when multiple (NTCs) are included in each assay to detect contamination
dilutions of the same sample are prepared. Cross- introduced during reaction setup. NTC qPCRs should
contamination between standards and/or samples will return Cq scores that are at least 3 cycles later than the
affect the accuracy of quantification. average Cq score for Standard 6.
• Avoid placing the pipette tip too far under the reagent Always dispense the DNA Standards from the lowest to the
surface during aspiration, as this may result in liquid highest concentration (i.e., from DNA Standard 6 to DNA
adhering to the outside of the tip. Standard 1) and use a fresh tip for each DNA Standard.
• After aspirating the desired volume of any reaction Melt curve analysis of NTC reactions may be performed
component, examine the pipette tip before dispensing to confirm whether amplification is due to contamination
to ensure that the correct volume is being transferred. with DNA Standard or library DNA, or due to primer-dimer
formation.
• Always try to dispense reaction components as close
as possible to the bottom of the tube or well. Primer-dimer formation is not uncommon with this
assay. This is due to primer design, which is not optimal
• Flush/rinse pipette tips by pipetting up and down 2 – 3 for qPCR, but dictated by the Illumina flow cell oligo
times after dispensing. sequences. The cycling times used are also much longer
• Ensure that no residual liquid remains in the tip after than those used in typical qPCR with KAPA SYBR® FAST,
dispensing. further increasing chances of primer-dimer formation.
As long as NTC amplification is at least 3 cycles after
Sample Concentration and Dilutions DNA Standard 6, primer-dimer formation should have no
impact on kit performance.
Libraries and controls must be diluted to fall within the
dynamic range of the assay, i.e., 20 – 0.0002 pM or Reaction Volume
5.5 – 0.000055 pg/µL or 12 x 106 – 12 x 101 dsDNA While this protocol specifies 20 µL reactions, volumes
molecules/µL. Any library dilution that amplifies before may be scaled down to 10 µL, if required. For improved
DNA Standard 1 should not be used in library concentration accuracy, the volume of DNA Standards/library dilutions
calculations. If only one dilution was included in the assay,

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

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
should be kept at 4 µL, with 6 µL of KAPA SYBR® FAST • An existing, previously sequenced library is a valuable
qPCR Master Mix with Primer Premix. However, the internal control, as both qPCR-based concentration
amount of template (DNA Standard or diluted library) used and cluster density data will be available for such a
per reaction may be scaled as required, provided that control. The biggest risk of this control is degradation
it can still be pipetted accurately. Always use the same of DNA quality over time, particularly if the same library
volume of DNA Standard and diluted library. Ensure that is used repeatedly as an internal control. The best
plastic consumables, pipettes, and qPCR instruments are approach is to select one or more internal controls from
compatible with the reaction volume. a pool of recently prepared and sequenced libraries,
which have been stored in a buffered solution at -20ºC,
Internal Controls
and have not been subjected to too many freeze-thaw
The dilution of concentrated library DNA to fall within the cycles. Single-use aliquots of libraries can be prepared
dynamic range of this assay represents the biggest risk and stored at -20ºC for use as controls.
to accurate quantification, particularly if libraries are very
concentrated and large initial dilutions are required. • The use of PhiX as an internal library quantification
control has similar advantages as that of a previously
If more than one dilution of each library is assayed (and sequenced library. However, PhiX is not recommended
falls within the dynamic range of the standard curve), the if only one internal control is included in a library
ΔCq value for consecutive dilutions is a good indication quantification assay, due to reported batch-to-batch
of the reliability of calculated library concentrations as in variation in the given concentration and average
Working Example (step 5). However, ΔCq values for serial fragment length of different lots.
dilutions of a library do not provide any indication of the
accuracy of the initial dilution. Replicates, Data Reliability, Throughput,
For this reason, we recommend including at least one and Per-sample Cost
appropriate internal process or dilution control in every qPCR is an extremely sensitive measurement technique
assay. These include: that is vulnerable to variation arising from a number of
sources. Triplicate qPCRs are recommended for DNA
• KAPA Library Quantification Dilution Control (KK4906).
Standards, library samples, and controls.
This is a quality-controlled 200 pM solution of the same
linear, 452 bp dsDNA fragment comprising the DNA The number of replicates may be reduced to two in
Standards and is also referred to as DNA Standard 0. order to increase throughput and reduce per-sample
cost. When selecting the best strategy for your workflow
• An Illumina library that has previously been quantified
and throughput requirements, keep in mind that the
with the KAPA Library Quantification Kit and that has
reliability of data is inversely proportional to the number
been sequenced successfully.
of replicates. Reducing the number of replicates increases
• PhiX, a control library supplied by Illumina. the risk of having to re-assay libraries if reliable data was
not obtained.
To be most effective, the internal control should be
The risk of reducing the number of replicate qPCRs can
processed in the same way as the libraries to be assayed,
be mitigated by designing workflows in such a way that
i.e., the same initial dilution and serial dilutions should be
at least two serial dilutions of each library are always
prepared, and replicate reactions set up with each dilution
assayed, provided that both of these dilutions fall within
of the internal control. Each of the internal controls listed
the dynamic range of the assay.
above have advantages and disadvantages:
For high-throughput library construction pipelines,
• The KAPA Library Quantification Dilution Control (DNA
automated library quantification in 384-well format is highly
Standard 0) is subject to the same rigorous quality
recommended, as this offers the possibility of quantifying
control as the set of DNA Standards supplied in the
96 libraries in triplicate in a single run, while reducing the
KAPA Library Quantification Kit. Absolute concentration
per-sample cost by performing 10 µL qPCRs.
and minimal lot-to-lot variation is guaranteed. At
200 pM, however, DNA Standard 0 is more dilute than Assay Automation
most Illumina libraries. If DNA Standard 0 is diluted to Library quantification with the KAPA Library Quantification
the same extent as the samples to be assayed, the Cq Kit is amenable to automation and the use of automated
scores for the dilutions are therefore likely to be a few liquid handling platforms is highly recommended for high-
cycles higher than for the libraries. This is acceptable, throughput library quantification workflows.
as long as at least one of the dilutions of Standard 0
falls within the dynamic range of the assay. Please note Pre-validated KAPA Library Quantification methods are
that the KAPA Library Quantification Dilution Control available from selected suppliers of automated liquid
(DNA Standard 0) is not suitable as a sequencing handling platforms. For more information, please contact
control, as it is a homogenous solution of a single Technical Support at kapabiosystems.com/support.
species of dsDNA and not a library.

4 For Research Use Only. Not for use in diagnostic procedures.

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Process Workflow

Combine:
5 mL KAPA SYBR FAST qPCR Master Mix (2X)
® Record date and lot numbers of reagents used.
Reagent Preparation Store at -20°C and protect from light.
1 mL Primer Premix (10X) Minimize freeze-thaw cycles.
0.2 mL ROX High or Low (50X) (optional)

Dilute library DNA and any internal Prepare fresh dilutions for each assay
controls with library dilution buffer
Sample Preparation Keep diluted DNA on ice
[10 mM Tris-HCl, pH 8.0 – 8.5
(25°C) + 0.05% Tween® 20 (optional)] to prevent degradation

Per 20 µL reaction:
12 µL of KAPA SYBR FAST + Primer Premix*
Reactions may be scaled down to 10 µL
4 µL of each DNA Standard/library dilution
Include no-template control reactions
Reaction Setup
PCR-grade water up to 20 µL
Work from lowest to highest DNA Standard
*If ROX is included, use 12.4 µL per 20 µL to avoid cross-contamination
reaction. ROX can also be added separately
(0.4 µL per reaction)

95°C – 5 min
35 cycles of 95°C – 30 sec Increase combined annealing/extension
qPCR Cycling to 90 sec for libraries with average
60°C – 45 sec [data acquisition] fragment length >700 bp.
Melt: 65 – 95°C (optional)

Remove outliers Absolute quantification against standard

curve (20 pM – 0.2 fM)
Confirm reaction efficiency 90 – 110%
Data Analysis Use the KAPA Library Quantification
Confirm R2 >0.99
Data Analysis Template designed for library
Confirm ΔCq for DNA Standards 3.1 – 3.6 quantification data analysis

Confirm correct ΔCq for library dilutions

Data exported from qPCR instrument
Confirm library dilutions amplify within
dynamic range of assay Library Concentration Calculation Library concentration =
qPCR conc x (452 bp/average fragment
Perform size-adjustment calculation
length) x dilution factor
Calculate undiluted library concentration

For Research Use Only. Not for use in diagnostic procedures. 5

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Detailed Protocol 3. Reaction Setup and Cycling
1. Reagent Preparation 3.1 D
etermine the total number of reactions that
will be performed for the appropriate number of
1.1 P
 repare an appropriate volume of DNA dilution replicates of each of the following reactions:
buffer [10 mM Tris-HCl, pH 8.0 – 8.5 (25°C) +
0.05% Tween® 20 (optional)]. This buffer can be • six DNA Standards
stored at room temperature or 4°C and re-used. • each dilution of every library to be assayed
Always equilibrate the buffer to room temperature
before use. • each dilution of every internal control
1.2 E
 nsure that all components of the KAPA Library • no-template controls (NTCs)
Quantification Kit are completely thawed and
thoroughly mixed. 3.2 P
 repare the required volume of master mix using
the reaction setup recommended below.
1.3 If the kit is used for the first time, add the Primer
Reaction setup: 20 µL reactions
Premix (10X) (1 mL) to the bottle of KAPA SYBR®
FAST qPCR Master Mix (2X) (5 mL). Mix thoroughly For Universal qPCR Master Mix ROX No ROX
using a vortex mixer. KAPA SYBR FAST qPCR Master
12.0 µL 12.0 µL
If you are using a Universal qPCR Master Mix Mix (2X) + Primer Premix (10X)1
kit and will only use ROX High or ROX Low, the ROX High or Low (50X)
0.4 µL 0 µL
appropriate ROX solution (50X) (0.2 mL) may be (see Table 1)
added to the qPCR Master Mix with primers when PCR-grade water 3.6 µL 4.0 µL
the kit is first opened. The volume of this mixture Total volume: 16.0 µL 16.0 µL
used per reaction should be adjusted accordingly If ROX was added to the qPCR Master Mix and primers, use 12.4 µL of
1

(12.4 µL per 20 µL reaction or 6.2 µL per 10 µL the qPCR Master Mix with Primer Premix and ROX per reaction.
reaction).
For ABI Prism™, Bio-Rad iCycler™, LightCycler
1.4 R
 ecord the lot numbers of all reagents, as well
480 or ROX Low qPCR Master Mix
as the date on which the primers (and ROX) were
KAPA SYBR FAST qPCR Master
added to the qPCR Master Mix. KAPA SYBR FAST 12.0 µL
Mix (2X) + Primer Premix (10X)
qPCR Master Mixes with primers (and ROX) are
stable through 30 freeze-thaw cycles, and should PCR-grade water 4.0 µL
be stored protected from light at -20°C when not Total volume: 16.0 µL
in use. Mixes may be stored in the dark at 4°C for
≤1 week, provided that they are not contaminated Reaction setup: 10 µL reactions
with microbes and/or nucleases during preparation For Universal qPCR Master Mix ROX No ROX
or subsequent use in reaction setup. KAPA SYBR® FAST qPCR Master
6.0 µL 6.0 µL
Mix (2X) + Primer Premix (10X)1
2. Sample Preparation 50X ROX High or Low
0.2 µL 0 µL
2.1 P
 repare the appropriate library dilutions (using (see Table 1)
DNA dilution buffer). Depending on the expected Total volume: 6.2 µL 6.0 µL
concentration of the library, 1:1,000 – 1:100,000 1
If ROX was added to the qPCR Master Mix and primers, use 6.2 µL of
dilutions may be appropriate. At least one additional the qPCR Master Mix with Primer Premix and ROX per reaction.
2
 he recommended reaction setup results in a total reaction volume of
T
2-fold dilution of each library is recommended. 10.2 µL if ROX is added during reaction setup. This does not impact
performance.
2.2 Prepare the required internal control dilutions.

 able 1. Recommended ROX concentrations for use

T For ABI Prism™, Bio-Rad iCycler™,
with KAPA SYBR FAST Universal qPCR Master Mix LightCycler® 480 or ROX Low qPCR Master Mix

Instrument ROX KAPA SYBR FAST qPCR Master Mix (2X) 6.0 µL
+ Primer Premix (10X)
Applied Biosystems® 5700, 7000, 7300, 7700, ROX
7900HT, StepOne™, and StepOnePlus™ High Total volume: 6.0 µL

Applied Biosystems 7500, ViiA™7,

QuantStudio™ 12K Flex, Agilent
ROX 3.3 Mix and briefly centrifuge the reagent master mix.
Low
Mx3000P™, Mx3005P™, and Mx4000™ 3.4 D
 ispense the appropriate volume of the master
Rotor-Gene™, DNA Engine Opticon™, mix into each PCR tube or well.
Opticon™ 2, Chromo 4™ Real-Time
Detector, Mastercycler® ep realplex, Smart No 3.5 Add 4 µL of PCR-grade water to all NTC well/tube(s).
Cycler®, Roche LightCycler® 480, Roche ROX
LightCycler Nano, Bio-Rad CFX96, and
Illumina Eco™

6 For Research Use Only. Not for use in diagnostic procedures.

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
3.6 D
 ispense 4 µL of each DNA Standard into the a library fall outside the standard curve, re-quantify
appropriate well/tube(s), working from the most a more appropriate dilution of the library.
dilute (Standard 6) to the most concentrated
4.4 U
se the instrument software to generate the
(Standard 1).
standard curve. The standard curve may also
3.7 D
 ispense 4 µL of each dilution of libraries and be generated manually using the KAPA Library
internal controls to be assayed. Quantification Data Analysis Template.
3.8 C
 ap tubes or seal the PCR plate, and transfer to 4.5 R
 eview the standard curve to ensure that the
the qPCR instrument. following criteria are met:
3.9 P
 erform qPCR with the following cycling protocol, • The average ΔCq value between DNA
selecting the Absolute Quantification option in Standards is in the range of 3.1 – 3.6.
the instrument software. Adjust run parameters
• The calculated reaction efficiency is in the
(e.g., reporters, reference dyes, gain settings, etc.)
range of 90 – 110% (i.e., the PCR product has
as required.
increased 1.8- to 2.2-fold per cycle, and the
Step Temp. Duration Cycles slope of the standard curve is between -3.1
and -3.6).
Initial denaturation 95ºC 5 min 1
Denaturation 95ºC 30 sec • R2 ≥0.99.
Annealing/Extension/ 35
60ºC 45 sec1 If the standard curve does not meet these criteria,
Data acquisition
calculated library concentrations will not be
Melt curve analysis2 65 – 95°C
reliable, and the assay must be repeated.
Increase to 90 sec for long-insert libraries (>700 bp).
1

Optional; see Data Analysis and Interpretation (p. 9) for more details.
2 4.6 M
 ost qPCR software will calculate the concentration
of the library and control dilutions using absolute
4. Data Analysis quantification against the standard curve. However,
we recommend exporting qPCR data to the KAPA
NOTE: A working example of library quantification
Library Quantification Data Analysis Template to
data analysis is provided in Working Example
perform the following calculations to determine the
(step 5).
undiluted library concentration:
4.1 A
 nnotate the DNA Standards as outlined below.
• Use the standard curve to convert the average
Note that the specified values correspond to the
Cq score for each dilution of every library and
concentrations of the DNA Standards, and not
internal control that was assayed to average
the final DNA concentration in each reaction.
concentration (in pM).
It is not necessary to convert these to the actual
concentrations in the reaction, as long as the same • Calculate the average size-adjusted
volume of template (DNA Standard, diluted library concentration (in pM) for each dilution of
or internal control) is used in all reactions. every library and control that was assayed,
by multiplying the calculated average
DNA Standard 1 20 pM concentration with the following factor:
DNA Standard 2 2 pM
Size of DNA Standard in bp (452)
DNA Standard 3 0.2 pM
DNA Standard 4 0.02 pM Average fragment length of library in bp
DNA Standard 5 0.002 pM Please refer to Data Analysis and Interpretation
DNA Standard 6 0.0002 pM (p. 9) for more information about the size-
adjustment calculation.
4.2 R
 eview the background-subtracted (normalized) • Multiply the average size-adjusted
amplification curves and the Cq scores for concentration calculated for each dilution of
replicate data points (DNA Standards, libraries and every library or control that was assayed with
controls), and exclude obvious outliers. Replicate the appropriate dilution factor to calculate
data points should differ by ≤0.2 cycles. If the data final concentration for the undiluted library or
set contains many outliers, results are unlikely to control from each of the dilutions assayed.
be reliable. Repeat the assay with particular focus
on improving pipetting accuracy. 4.7 R
 eview the final calculated concentrations and
determine the working concentration for each
4.3 E
 xclude all library dilutions that fall outside the
sample to be used for downstream processing
dynamic range of the assay, i.e., that return an
(e.g., pooling for target enrichment or cluster
average Cq score lower than that of Standard 1 or
amplification).
higher than that of Standard 6. If all the dilutions of

For Research Use Only. Not for use in diagnostic procedures. 7

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
5. Working Example After excluding outliers (>0.2 Ct difference) for
DNA Standards 2 and 6, the standard curve was
Three indexed DNA libraries were prepared from
generated (Figure 1). The quality control metrics
250 ng of Covaris-sheared human genomic DNA
for the standard curve were as follows:
using the KAPA LTP Library Preparation Kit, for
2 x 100 bp paired-end whole-genome shotgun • ΔCq for all pairs of DNA Standards was within
sequencing on the Illumina HiSeq® 2500. Adapter- the specified range of 3.1 – 3.6.
ligated libraries were size-selected (250 – 450 bp)
• The reaction efficiency of 95% was within the
and amplified (6 cycles). Amplified libraries were
specified range of 90 – 110%.
analyzed using an Agilent Bioanalyzer High
Sensitivity DNA Assay to determine the average • The R2 value of 0.9999 met the specification
fragment size and approximate concentration of of ≥0.99.
each library (Table 2, rows 1 and 2).
An initial 1:10,000 dilution and one additional 2-fold
(i.e., 1:20,000) dilution of each library was prepared.
As a process control, 1:10,000 and 1:20,000
dilutions of the KAPA Library Quantification Internal
Control (Illumina DNA Standard 0, 200 pM) were
also made. Samples were assayed using the KAPA
Library Quantification Kit. All DNA Standards and
library dilutions were assayed in triplicate, and
triplicate NTCs were included.
The triplicate and average Cq scores for the
six DNA Standards and NTCs are given in the Figure 1. Standard curve generated with the KAPA Library Quantification Kit for
following table. Illumina sequencing platforms

DNA Conc Cq Average Triplicate Cq scores for each dilution of the three
ΔCq*
Standard (pM) score Cq libraries and internal control are given in Table 2,
7.15 row 4. Outliers (>0.2 Ct difference) for the 1:20,000
1 20 7.22 7.20 – dilution of Library 1 and the 1:10,000 dilution of
7.23 Library 3 were excluded before the average Cq
score (row 5) was calculated for each sample.
10.63
2 2 10.90 10.66 3.46 For an indication of the reliability of concentrations
10.69
to be calculated, the ΔCq for the two dilutions of
each library and the internal control was calculated.
14.13
For consecutive 2-fold dilutions of a sample, a
3 0.2 14.12 14.13 3.47 ΔCq of 1.0 is expected, and values between 0.9
14.13 and 1.1 are acceptable. This indicates that the
17.72 concentration of a library calculated from two or
4 0.02 17.65 17.68 3.55
more dilutions is likely to differ by <10%. If the ΔCq
values for consecutive dilutions of a sample fall
17.66
outside the acceptable range, indications are that
21.01 quantification may not be reliable, as a result of
5 0.002 21.14 21.08 3.40 poor liquid handling and/or sample contamination.
21.08 Next, the average Cq score for each library/
25.00 control dilution was converted to concentration
6 0.0002 24.45 24.44 3.36 (in pM) using the standard curve (Table 2, row 7).
24.42 The average size-adjusted concentration of each
library dilution was subsequently calculated (as
34.5
outlined in Detailed Protocol (step 4.6); Table 2,
NTC N/A ND >34 N/A row 8). Finally, the average final concentration
33.9 (in nM) of each undiluted library and the control
*Should be 3.1 – 3.6 for a 10-fold template dilution series. was calculated from each of the dilutions that
were assayed (Table 2, row 9). The calculated
concentration of the internal control (205.4 pM
and 207.8 pM, from the 1:10,000 and 1:20,000
dilutions, respectively) was within 10% of the given

8 For Research Use Only. Not for use in diagnostic procedures.

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Table 2. Working example of qPCR-based quantification of libraries prepared for Illumina sequencing

Row Parameter Library 1 Library 2 Library 3 Std 0 control

1 Average fragment length (Bioanalyzer) 340 bp 335 bp 351 bp 452 bp
17 ng/µL 18 ng/µL 27 ng/µL 200 pM
2 Estimated concentration (Bioanalyzer)
= 80.9 nM = 87.0 nM = 124.5 nM (as supplied)
3 Dilutions for qPCR 1:10K 1:20K 1:10K 1:20K 1:10K 1:20K 1:10K 1:20K
9.24 10.21 8.79 9.79 8.30 9.40 17.50 18.53
4 Triplicate Cq scores 9.25 10.12 8.84 9.81 8.65 9.22 17.58 18.62
9.21 10.53 8.88 9.94 8.34 9.26 17.57 18.57
5 Average Cq score 9.23 10.17 8.84 9.85 8.32 9.29 17.55 18.57
6 ΔCq 0.93 1.01 0.97 1.02
Average concentration for sample dilution
7 5.23 2.81 6.82 3.48 9.62 5.03 0.021 0.010
calculated using standard curve (pM)
Average size-adjusted concentration for
8 6.96 3.74 9.20 4.69 12.39 6.48 0.021 0.010
library dilution (pM)
Average final calculated concentration of
9 69.6 74.8 92.0 93.9 123.9 129.5 0.205 0.208
undiluted library dilution (nM)
Deviation between final concentrations
10 7.5% 2.1% 4.6% 1.2%
calculated from different dilutions
72.2 nM 92.9 nM 126.7 nM
11 Working concentration (206.6 pM)
= 15.2 ng/µL = 19.2 ng/µL = 27.5 ng/µL

value, indicating that no gross errors occurred Data Analysis and Interpretation
during sample dilution, or setup and execution of
the assay. Early Amplification of DNA Standard 1
The concentration of DNA Standard 1 is much higher than
To calculate the working concentration of each that of samples that are typically analyzed by qPCR. As
library, the variation between the two different a result, DNA Standard 1 is amplified at a very early Cq.
dilutions was determined. Since calculated Most qPCR instruments establish a signal baseline during
concentrations varied by <10% for all libraries, the the first 3 – 15 cycles of the qPCR, and any increase
average value of the two dilutions was selected as in fluorescence during those cycles is interpreted as
the working concentration. If the values differ by background. This can result in significant problems with
>10%, working concentrations should be based the standard curve, since Standard 1 can be interpreted
on one of the following: purely as background or its Cq score can be somewhat
• The average value from dilutions that returned delayed. To avoid problems with DNA Standard 1, the
final calculated concentration values that cycles used for baseline/background subtraction should
varied <10%. be manually adjusted to cycles 1 – 3.
• The final calculated concentration for the Size-adjustment Calculation
lowest dilution of each library that appears to Size-adjustment must be performed to compensate for the
be reliable. difference in average fragment length between the library
that was assayed and the DNA Standard. The fluorescent
signal generated by SYBR® Green I is dependent on the
total mass of DNA. Therefore, a longer amplicon at a lower
concentration may produce the same amount of signal as
a shorter amplicon at a higher concentration.
The size-adjustment calculation is a simple multiplication
of the concentration derived from the standard curve with
the ratio between the size of the DNA Standard (452 bp)
and the average fragment size for that particular library
(see Detailed Protocol and Working Example).

For Research Use Only. Not for use in diagnostic procedures. 9

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Melt Curve Analysis • qPCR-based quantification with the engineered KAPA
Melt curve analysis may be useful for the identification SYBR FAST DNA Polymerase ensures that all the
of carry-over adapter-dimer in Illumina libraries (Figure 2) molecules in a complex DNA population are quantified
and/or to identify contamination. The melt curves for the with high efficiency, irrespective of fragment length
KAPA Library Quantification DNA Standard for Illumina or GC content. Cluster amplification (bridge PCR) is,
platforms displays a characteristic double peak. This is however, performed with a different enzyme and is
the result of differential local melting in the 452 bp linear more challenging. Cluster generation may therefore not
template and is not indicative of non-specific amplification. be equally efficient for all library fragments.
• Anecdotal evidence suggests that not all library types
cluster similarly on a specific instrument and libraries
of the same type do not yield the same number of
clusters on different instruments of the same type
when loaded at a specific concentration. Reasons
for this fall outside the scope of this document.
The SeqAnswers blog is an excellent resource for
insightful NGS community forum discussions.
Ultimately, the correlation between library concentration
determined with the KAPA Library Quantification Kit and
cluster density must be determined empirically for your
libraries, instrumentation, and workflows. To discover
and define this correlation, it is important that the assay
is performed meticulously and that data analysis and
interpretation is performed correctly. If you are new to
NGS and qPCR-based quantification, the following may
be helpful:
Figure 2. Melt curve analysis of libraries and DNA Standards amplified with the
KAPA Library Quantification Kit for Illumina platforms. Libraries in the top panel • Assay different dilutions of a few libraries (preferably
contain no or a negligible amount of adapter-dimer. Libraries in the bottom panel previously sequenced) on different days, to measure
contain an unacceptable amount of adapter-dimer, contributing to an inflated
calculated library concentration. Note the characteristic double melt peak for the the reproducibility of qPCR data obtained. As indicated
DNA Standard. previously, fresh dilutions of test libraries used for this
Correlation between Library Concentration and evaluation should be prepared for each assay. Variation
Cluster Density of ≤10% in the concentration of a library calculated
from different dilutions (within an assay) or in separate
The KAPA Library Quantification Kit has become an
assays, is generally regarded as acceptable.
integral part of Illumina sequencing workflows, in both
low- and high-throughput environments. The assay is • Perform cluster amplification with the same set of test
capable of yielding reproducible, accurate, and reliable libraries, more than once, on the instrument(s) that will
results. The guidelines and instructions provided in this be used routinely. This is important to determine the
document, particularly in Data Analysis (step 4) and variability of the cluster amplification process.
Working Example (step 5), should enable both new and
• Establish and maintain a database of library
experienced users to determine whether each data set
concentrations (determined with the KAPA Library
generated with the assay is reliable. Since the metrics
Quantification Kit and any other quantification
used to evaluate data cannot rule out gross errors in
methods that are being used), as well as cluster
library dilutions, calculated library concentrations can
density data generated for different library types and/
only be accepted with confidence if an internal, process
or instruments. Data will enable you to define the
or dilution control was included in the assay.
correlation between calculated library concentration
Even if the result generated with the assay is determined and cluster density for your specific situation and will
to be reliable, there is no guarantee that libraries diluted for also be invaluable for process optimization, quality
flow cell loading will yield an optimal number of clusters. control, and troubleshooting.
Reasons for possible discrepancies between calculated
library concentration and cluster density include:
• There are multiple liquid handling steps between library
quantification and flow cell loading, all of which are
prone to error.
• Cluster amplification is a complex process with many
variables, some of which are instrument-related.

10 For Research Use Only. Not for use in diagnostic procedures.

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
Comparison with Other Quantification Methods In over-amplified libraries, a large proportion of the DNA may
Most NGS workflows employ more than one library be partially single-stranded due to substrate depletion and
quantification method. Data generated with a spectro- subsequent formation of heteroduplex molecules, which
photometer (e.g., NanoDrop™), a fluorometric leads to severe underestimation of library concentrations
(e.g., Qubit® or PicoGreen®) assay, or an electrophoretic when dsDNA-binding dyes are used. Conversely, library
device (e.g., a Bioanalyzer, TapeStation or LabChip® GX) quantification by qPCR, involves denaturation of the
may be used to determine the most appropriate initial entire library to single-stranded form, allowing accurate
dilutions for accurate, qPCR-based quantification using quantification of all PCR-competent molecules.
the KAPA Library Quantification Kit. Theoretically, library A detailed explanation and supporting data may be found
concentrations determined with the Library Quantification in the KAPA Library Quantification Technical Guide—
Kit should always be similar to or lower than concentrations available upon request at kapabiosystems.com/support.
determined with spectrophotometric methods, which
quantify total DNA, since qPCR measures only those
molecules that can be amplified in PCR. However, when
quantifying very concentrated or overamplified libraries,
this may not be the case.

Troubleshooting
Symptom Possible causes
•E
 fficiency above 100% may be indicative of contamination, where Standard 6 is <3.1 cycles from
Efficiency not in Standard 5, and NTC reactions amplify less than 3 cycles from Standard 6. Examine melt curves to
specified range determine the source of contamination (Standard DNA or library DNA).
(90 – 110%) •B
 aseline setting may delay Cq score for Standard 1, affecting efficiency. Adjust baseline manually.
•P
 oor liquid handling
•P
 oor liquid handling. Ensure that all reagents are thoroughly mixed before use.
Poor R2 value (<0.99)
• Instrument-related issues. Ensure that the correct reference dye was used, at the correct concentration.
•Δ
 Cq of <3.1 between DNA Standards 5 and 6 is indicative of contamination. Examine melt curves to
determine whether contaminant is Standard or library DNA.
•Δ
 Cq of <3.1 between DNA Standards 1 and 2 may be indicative of problems with background
Standards are not subtraction. Adjust the baseline manually.
spaced correctly •Δ
 Cq of >3.6 points to poor reaction efficiency. Ensure that all reagents are thoroughly mixed before use.
(3.1 – 3.6 cycles apart) Confirm that all reaction components were added at the correct concentration, and that the correct
cycling protocol was used.
 evere light exposure of KAPA SYBR® FAST qPCR Master Mix will reduce total fluorescence and may
•S
result in a delay of Cq scores, resulting in ΔCq values of >3.6.
Poor reproducibility • Poor liquid handling. Ensure that all reagents are thoroughly mixed before use.
between replicates • Instrument-related issues. Ensure that the correct reference dye was used, at the correct concentration.
ΔCq of library dilutions • Poor liquid handling in preparation of library dilutions.
is not within expected
• Library is challenging to amplify, i.e., extremely GC- or AT-rich, or has an average fragment length >1 kb.
range (0.9 – 1.1 for
2-fold dilutions) • Library DNA has degraded. Prepare fresh dilutions and keep on ice during reaction setup.
Concentrations • Poor liquid handling in preparation of library dilutions.
calculated from different
•L  ibrary is challenging to amplify, i.e., extremely GC- or AT-rich, or has an average fragment length >1 kb.
library dilutions differ by
more than 10% • Library DNA has degraded. Prepare fresh dilutions and keep on ice during reaction setup.
• Dilutions that amplify before DNA Standard 1 should not be used. Use only those dilutions that fall
Library dilutions do within the range of the standard curve. If no such dilutions were assayed, repeat the assay with a more
not fall within dynamic appropriate dilution factor.
range of standard curve • Libraries that amplify after Standard 6 are unlikely to contain a significant amount of library DNA. Repeat
the quantification to confirm the result.
Standard 1 amplification • Instrument is subtracting early amplification as background. Adjust the baseline settings to use cycles
plot appears abnormal before any signs of amplification (1 – 3).
Standards amplify, but
libraries do not, or very • Library does not contain the appropriate adapter sequences for quantification primers to bind to.
late amplification of • Gross error with initial dilution, or library DNA has degraded. Re-assay fresh dilutions.
libraries

For Research Use Only. Not for use in diagnostic procedures. 11

KAPA Library Quantification Kit
Illumina® Platforms Technical Data Sheet
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This technical data sheet is provided “as is” and Kapa Biosystems
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to perform any such applications. Users of this product may

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12 For Research Use Only. Not for use in diagnostic procedures.