Automated Library Construction
Using KAPA Library Preparation Kits
on the Agilent NGS Workstation
Yields High-Quality Libraries for
Whole-Genome Sequencing on theIllumina Platform
Application NoteAuthors

Abstract

Nguyet Kong, Kao Thao, Carol Huang,

A new method was developed to automate the KAPA HTP Library Preparation kit forand Bart C. Weimer

microbial whole genome sequencing. This method uses the Agilent NGS

Population Health and Reproduction

Workstation, consisting of the NGS Bravo liquid handling platform with its acces-Department, School of Veterinary

sories for heating, cooling, shaking, and magnetic bead manipulations in a 96-wellMedicine, University offormat. User intervention in multistep protocols is minimized through the use of

California-Davis, Davis, CA, USA

other components of the workstation such as the BenchCel 4R Microplate Handler

Maryke Appeland Labware MiniHub for labware storage and movement. This method has been

Kapa Biosystems, Inc.

developed on the Illumina platform and consists of three protocols: the first is for

Wilmington, MA USA

end repair to post-ligation cleanup; the second is used for library amplification
setup; and the third is for the post-amplification cleanup. The modular design

Stephen Lappin, Lisa Knapp, andLenore Kelly
Agilent Technologies, Inc.
Santa Clara, CA, USAprovides the end-user with the flexibility to complete library construction over twodays, and is suitable for the construction of high-quality libraries from bacteria ofvarious GC content. This combined solution produced a workflow that is suitable forlarge-scale sequencing projects such as the 100K Pathogen Genome Project.

low-bias NGS library amplification [2,3,4,5]. These reagentswere selected by the 100K Pathogen Genome Project for automated library construction due to the excellent sequence
results in the initial phase of the project using manualapproaches. However, increased capacity required automationof this protocol to meet the high-throughput demands. Thisapplication note details the automation of high quality
bacterial libraries that are reproducible and provide excellent
sequencing results using the KAPA HTP Library Preparation
Kit on the Agilent NGS Workstation for use in the 100K
Pathogen Genome Project.

Introduction
Reduced costs and higher throughput have rendered microbial
whole genome sequencing (WGS) accessible to many applications in infectious disease, food safety, and public health toproduce genomes on an unprecedented scale. The 100K
Pathogen Genome Project hosted at UC Davis and founded byAgilent Technologies, the FDA, and UC Davis, is a novel
public/private/government consortium to sequence 100,000
bacterial zoonotic and food-borne pathogens that are of significant importance to the public, using next generation
sequencing (NGS) technologies. The project has sufficientsequencing capacity to produce as many as 25,000 bacterial
genomes per year using automated workflows. This capacity
in sequencing created a challenge to establish an automatedlibrary construction workflow to fully enable the reliable androbust sequencing pipeline.

Materials and Methods
The 100K Pathogen Genome Project sample preparation workflow for multiplexed, paired-end (2 × 100 bp) Illumina sequencing libraries uses accepted methods for bacterial sequencing
projects (Figure 1). Collection of high molecular weight
genomic DNA is critical for successful library construction.
Optimization of the cell lysis and DNA isolation is described inprevious documentation [6]. High-throughput QC of the highmolecular weight genomic DNA using the Agilent 2200
TapeStation System is also documented elsewhere [7].

NGS library construction comprises repetitive processes,
making it amenable to automation. Automation offers many
advantages, including sample throughput, reduced hands-ontime, greater reproducibility, and improved process control.
However, the development of automated protocols is nottrivial. Optimal workflows require robust chemistry, robotics,
and meticulous optimization of many parameters to achieve
acceptable libraries that yield sequence data of quality similar
to those produced by skilled, experienced, and attentive
technicians in a low-throughput setting.

DNA extraction

The NGS Workstation platform offers reliable and reproducible automation of library preparation processes. With
minimal user-intervention to produce robust libraries, thegraphical user interface of the Agilent VWorks Automation
Control Software makes the system easy to operate, while theopen architecture of the software facilitates new method
development and customization to optimize specific steps ofthe protocol.

QC: DNA quality and quantity

Shear DNA

QC: fragmentation

Library construction

Library preparation kits from Kapa Biosystems offer robust
library construction methods for a range of DNA inputs(100 pg–5 µg) for a variety of sequencing applications, including WGS, targeted sequencing, ChIP-Seq, RNA-Seq, andMethyl-Seq. Reagents are formulated for optimal activity andstability, and exhibit excellent conversion rates of input DNA
to adapter-ligated libraries through the use of a highly optimized, automation-friendly protocol using beads [1]. Kits contain the engineered KAPA HiFi DNA Polymerase, which has
become widely accepted for high-efficiency, high-fidelity,

Library normalizationand pooling

Submit for sequencing
Figure 1.

2100K Pathogen Genome Project sample preparation workflow for
multiplexed, short-read Illumina sequencing.

To validate the automated method, DNA for NGS library construction was extracted from selected bacterial isolates withdifferent GC content. These microbes are listed in Table 1.
Organisms were lysed using the KAPA Express Extract Kit(KK7102) [8], after which the DNA was purified with a Qiagen
QIAamp DNA Mini Kit (51306) using the manufacturer’sinstructions [9]. Before shearing, the extracted DNA wasanalyzed using an Agilent 2200 TapeStation system with theGenomic DNA ScreenTape assay for integrity of highmolecular weight DNA [7,10,11]. Fragmented DNA was quantified with the method described by Jeannotte et al. [7] using anAgilent 2200 TapeStation system as the 96-well platehigh-throughput workflow for quantitation and sizing of gDNA
samples for library construction [9].

Table 1.

Fragmentation

End repair

Library Construction Metrics for Libraries Prepared from Different
Bacteria
Approximate
genome size(MB)GCcontent(%)

Average Finallibrarylibrary
size (bp) yield (ng)Lactococcus Positive

235430679

Listeria

Positive

238307949

VibrioNegative

5 (two41chromosomes)

304

198

Escherichia

Negative

551

347

361

Salmonella

Negative

552

299

287

Bacterium

Input DNA

Gram
reactionA-tailing

Adaptor ligation

Size selection

DNA was sheared in batches of 96 samples using microtubes
with the Covaris E220 Focused Ultrasonicator [12]. The fragmented DNA size was determined with the Agilent 2100
Bioanalyzer system and High Sensitivity DNA Kit, to confirm anormal size distribution around a ~300 bp peak.

Library amplification

The input into library construction with the KAPA HTP LibraryPreparation Kit (KK8234, Figure 2) was normalized to 1–5 µg
for all samples [13]. The standard KAPA protocol withdual-SPRI size selection after adapter-ligation was used toachieve libraries with a fragment distribution in the range of250–450 bp. Library amplification was done for eight cycles
using the KAPA HiFi HotStart ReadyMix, followed by a final 1X
SPRI bead cleanup step. The size distributions of amplified
libraries were confirmed to be in the range of 200–500 bp,using the Agilent 2100 Bioanalyzer system with the HighSensitivity DNA Kit [14,15]. Libraries were quantified with theqPCR-based KAPA Library Quantification Kit (KK4824) prior tonormalization and pooling for sequencing [16] at BGI@UCD(Sacramento, CA) on the Illumina HiSeq 2000.

Figure 2.

3

Detailed KAPA HTP Library Preparation protocol. The input intolibrary construction is fragmented DNA or cDNA. Each enzymatic
reaction is followed by a SPRI-bead cleanup (1.7X after EndRepair; 1.8X after A-Tailing, and two consecutive 1X cleanups
after adapter ligation). The “with-bead” protocol uses a single
aliquot of SPRI beads for all cleanups prior to library amplification,and significantly reduces the loss of library fragments associatedwith the physical transfer of material between enzymatic reactions. This results in higher yields of adapter-ligated libraries, andreduces the number of amplification cycles required to generatesufficient material for Library QC and sequencing.

The entire library construction workflow, from fragmentation
to the post-amplification cleanup, can be completed in as littleas 9 hours. The automated KAPA HTP Library Preparation
method for the NGS Workstation (p/n G5522A; Figure 3) consists of three protocols, each with a graphical user interface
or Form (Figure 4A–4C). Three variations of the first protocol(end repair to post-ligation cleanup) are available to provide
the end user with the option of preparing libraries without
size selection, employing a SPRI-bead based size selection, orperforming off-deck size selection. The second protocol isused for library amplification setup, and the third for thepost-amplification cleanup. This modular design provides theend-user with the flexibility to complete library construction
over two days, or implement physical separation of pre- andpost-PCR procedures. The VWorks Forms are supplemented
by a Microsoft Excel workbook (Figure 4D) to facilitate
reagent preparation and instrument setup, and can be used
to keep a complete record of each experiment.

Figure 3.

Consumables used on the NGS Workstation were selected foroptimal performance. Labware movements and pipetting parameters for the KAPA HTP Library Preparation protocols were
carefully optimized to minimize reagent and consumablewaste and ensure equal or better library yields and quality
compared to manual preparation methods.

4The Agilent NGS Workstation. This instrument comprises thehighly reliable Agilent Bravo Automated Liquid Handling Platform,
Agilent Bravo accessories for heating, cooling, shaking, and magnetic bead manipulations. User intervention in multistep protocols
is minimized through the use of the BenchCel 4R Microplate
Handler and Labware MiniHub for labware storage and movement.

ABCD

Figure 4.

VWorks Forms and a Microsoft Excel workbook for the KAPA HTP Library Preparation method. A) End Repair to Post-ligation Cleanup Form, B) LibraryAmplification Setup Form, C) Post-amplification Cleanup Form, D) Microsoft Excel workbook. Each run is configured by selecting the number of platesample columns (1, 2, 3, 4, 6, or 12 columns, and 8, 16, 24, 32, 48, or 96 samples), after which, the interface displays the deck setup and workstation
setup for the protocol. The Microsoft Excel workbook is designed to guide reagent preparation and instrument setup, and can be used to create arecord of each experiment. The VWorks software has many additional, convenient features, including various ways of tracking the progress during arun, and intuitive manual control of over instrument components to facilitate error recovery.

5

different apparent library fragment sizes, but all of theselibraries produced adequate sequence results. The higher
molecular weight peak in the electropherograms for Listeria
libraries are typical of over-amplification (primer depletion
during library amplification). Since the average yield ofadapter-ligated library was higher for Listeria than for theother bacteria, the number of amplification cycles could havebeen reduced. The enteric pathogens also produced similar
sized libraries that produced excellent sequence results.

Results and Discussion
Prior to shearing, the extracted DNA was analyzed using theAgilent 2200 TapeStation system with the Genomic DNA
ScreenTape assay (Figure 5). A typical electropherogram forfragmented bacterial DNA used for library construction isgiven in Figure 6. Electropherograms and virtual gel images
for representative libraries prepared from Salmonella,Escherichia, Vibrio, Listeria and Lactococcus isolates using
the KAPA HTP Library Preparation method on an Agilent NGS
Workstation are given in Figure 7. Interestingly, libraries
generated from different lactococcal isolates displayed

Library construction metrics (average library size and final
library yields) for libraries prepared from different bacteria aresummarized in Table 1.

600

500

Sample intensity [FU]400300200Figure 5.

48,500

7,000

4,000

2,500
3,000

2,000

1,200
1,500

900

600

015,000

100[bp]Typical electropherogram generated with the Agilent 2200
TapeStation system and Genomic DNA ScreenTape assay toassess integrity of high molecular weight genomic DNA used forlibrary construction. Each line represents an individual Listeria
isolate.
D1[FU]
250
200
150
100
50
035Figure 6.

100 150 200300400500 600

1,000 2,000

10,380[bp]

A typical electropherogram (generated on the Agilent 2100 Bioanalyzer system andHigh Sensitivity DNA Kit) of Covaris-sheared DNA used for library construction.
Fragmentation parameters were selected to produce a fragment size distribution witha peak in the range of ~300 bp, which is ideal for 2 x 100 bp paired-end sequencing on
the Illumina HiSeq 2000.

6300250[bp] Ladder

200

7,000
2,000
1,000600
500
400
300
200
150
10035150
10050
0405060708090100

110120Lactococcus, 35% GC
The average library size for
Lactococcus is 430 bp

130 sec

300
250[bp] Ladder

200
150
10050
04050

60

70

80

90100110120

130 sec

7,000
2,000
1,000600
500
400
300
200
150
10035

Listeria, 38% GC
The average library size for
Listeria is 307 bp.

200[bp] Ladder

150
10050
0405060708090100

110120

sec

7,000
2,000
1,000600
500
400
300
200
150
100
35Vibrio, 41% GC
The average library size forVibrio is 304 bp.

200[bp] Ladder

150
7,000
2,000
1,000600
500
400
300
200
150
100
35

100
50
04050

60

70

80

90

100

110

120130sec

Escherichia, 51% GC
The average library size for
Escherichia is 347 bp.

250[bp] Ladder

200
7,000
2,000
1,000
600
500
400
300
200
150
100
35150
100
50
040Figure 7.

5060708090100

110120130

Salmonella, 52% GC
The average library size for
Salmonella is 299 bpsecRepresentative electropherograms and virtual gel images (generated on the Agilent 2100 Bioanalyzer system with the High Sensitivity DNA Kit) ofbacterial libraries prepared for whole genome sequencing with the KAPA HTP Library Preparation Kit on the Agilent NGS Workstation. The averagelibrary size for each genus was as indicated. Peaks at 35 and 10381 bp are internal standards used for alignment and quantitation determination withthe Agilent 2100 Bioanalyzer system.

7

Conclusion
The KAPA HTP Library Preparation Kit and Agilent NGSWorkstation provide a robust, high-throughput automation
solution for the construction of high-quality libraries for
microbial whole genome sequencing on the Illumina platform. This combined solution produced a workflow that issuitable for the construction of high-quality libraries for
microbial whole genome sequencing like the 100K Pathogen
Genome Project.

Acknowledgements
We gratefully acknowledge the technical assistance provided
by Winnie Ng, Poyin Chen, Narine Arabyan, Soraya Foutouhi,
Elizabeth Marta, Kerry Le, Sum Leung, Lucy Cai, Alan Truong,Christina Kong, Vivian Lee, Alvin Leonardo, Alex Weimer,Giovanni Tenorio, Patrick Ancheta, Azarene Foutouhi, andKendra Liu from the laboratory of Dr. Bart Weimer atUniversity of California, Davis.

References

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11. Agilent Genomic DNA ScreenTape System Quick Guide,
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Amplification/Illumina series (96 libraries):
http://www.kapabiosystems.com/products/name/kap
a-library-preparation-kits
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Technologies (p/n G2938-90321).
16. KAPA Library Quantification Kit - Illumina/Universal:
http://www.kapabiosystems.com/products/name/kap
a-library-quant-kits

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S. Fisher, et al., Genome Biology 12, R1 (2011).

For More Information

2.

M.A. Quail, et al., Nature Methods 9, 10–11 (2012).

3.

S.O. Oyola, et al., BMC Genomics 13, 1 (2012).

These data represent typical results. For more informationon our products and services, visit our Web site atwww.agilent.com/chem.

4.

M.A. Quail, et al., BMC Genomics 13: 341 (2012).

5.

M.G. Ross, et al., Genome Biology 14: R51 (2013).

6.

N. Kong, et al. Agilent Technologies Application Note(5991-3722EN): Production and Analysis of High
Molecular Weight Genomic DNA for NGS Pipelines
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7.

R. Jeannotte, et al. Agilent Technologies Application
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8.

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http://www.qiagen.com/products/catalog/sampletechnologies/dna-sample-technologies/genomicdna/qiaamp-dna-mini-kit

For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.

© Agilent Technologies, Inc., 2014, 2016
Printed in the USA
March 10, 2016
5991-4296EN