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

Abstract

A new method was developed to automate the KAPA HTP Library Preparation kit for microbial whole genome sequencing. This method uses the Agilent NGS Workstation, consisting of the NGS Bravo liquid handling platform with its accessories for heating, cooling, shaking, and magnetic bead manipulations in a 96-well format. User intervention in multistep protocols is minimized through the use of other components of the workstation such as the BenchCel 4R Microplate Handler and Labware MiniHub for labware storage and movement. This method has been validated for sequencing on the Illumina platform and consists of three protocols: the first is for 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 provides the end-user with the flexibility to complete library construction over two days, and is suitable for the construction of high-quality libraries from bacteria of various GC content. This combined solution produced a workflow that is suitable for production-scale sequencing projects such as the 100K Pathogen Genome Project.

Full text

Automated Library Construction
Using KAPA Library Preparation Kits
on the Agilent NGS Workstation
Yields High-Quality Libraries for
Whole-Genome Sequencing on the
Illumina Platform
Authors
Nguyet Kong, Kao Thao, Carol Huang,
and Bart C. Weimer
Population Health and Reproduction
Department, School of Veterinary
Medicine, University of
California-Davis, Davis, CA, USA
Maryke Appel
Kapa Biosystems, Inc.
Wilmington, MA USA
Stephen Lappin, Lisa Knapp, and
Lenore Kelly
Agilent Technologies, Inc.
Santa Clara, CA, USA
Application Note
Abstract
A new method was developed to automate the KAPA HTP Library Preparation kit for
microbial whole genome sequencing. This method uses the Agilent NGS
Workstation, consisting of the NGS Bravo liquid handling platform with its acces-
sories for heating, cooling, shaking, and magnetic bead manipulations in a 96-well
format. User intervention in multistep protocols is minimized through the use of
other components of the workstation such as the BenchCel 4R Microplate Handler
and Labware MiniHub for labware storage and movement. This method has been
validated for sequencing on the Illumina platform and consists of three protocols:
the first is for 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 provides the end-user with the flexibility to complete library construction
over two days, and is suitable for the construction of high-quality libraries from bac-
teria of various GC content. This combined solution produced a workflow that is
suitable for production-scale sequencing projects such as the 100K Pathogen
Genome Project.

2
Introduction
Reduced costs and higher throughput have rendered microbial
whole genome sequencing (WGS) accessible to many appli-
cations in infectious disease, food safety, and public health to
produce genomes on an unprecedented scale. The 100K
Pathogen Genome Project hosted at UC Davis and founded by
Agilent 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 sig-
nificant importance to the public, using next generation
sequencing (NGS) technologies. The project has sufficient
sequencing 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 automated
library construction workflow to fully enable the reliable and
robust sequencing pipeline.
NGS library construction comprises repetitive processes,
making it amenable to automation. Automation offers many
advantages, including sample throughput, reduced hands-on
time, greater reproducibility, and improved process control.
However, the development and validation of automated proto-
cols is not trivial. Optimal workflows require robust chemistry,
robotics, and meticulous optimization of many parameters to
achieve acceptable libraries that yield sequence data of qual-
ity similar to those produced by skilled, experienced, and
attentive technicians in a low-throughput setting.
The NGS Workstation platform offers reliable and repro-
ducible automation of library preparation processes. With
minimal user-intervention to produce robust libraries, the
graphical user interface of the Agilent VWorks Automation
Control Software makes the system easy to operate in a pro-
duction setting, while the open architecture of the software
facilitates new method development and customization to
optimize specific steps of the protocol.
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, includ-
ing WGS, targeted sequencing, ChIP-Seq, RNA-Seq, and
Methyl-Seq. Reagents are formulated for optimal activity and
stability, and exhibit excellent conversion rates of input DNA
to adapter-ligated libraries through the use of a highly opti-
mized, automation-friendly protocol using beads [1]. Kits con-
tain the engineered KAPA HiFi DNA Polymerase, which has
become widely accepted for high-efficiency, high-fidelity,
low-bias NGS library amplification [2,3,4,5]. These reagents
were selected by the 100K Pathogen Genome Project for auto-
mated library construction due to the excellent sequence
results in the initial phase of the project using manual
approaches. However, increased capacity required automation
of this protocol to meet the high-throughput demands. This
application note details the automated production 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.
Materials and Methods
The 100K Pathogen Genome Project sample preparation work-
flow for multiplexed, paired-end (2 × 100 bp) Illumina sequenc-
ing 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 in
previous documentation [6]. High-throughput QC of the high
molecular weight genomic DNA using the Agilent 2200
TapeStation System is also documented elsewhere [7].
Figure 1. 100K Pathogen Genome Project sample preparation workflow for
multiplexed, short-read Illumina sequencing.
DNA extraction
QC: DNA quality and quantity
Shear DNA
QC: fragmentation
Library construction
Library normalization
and pooling
Submit for sequencing

3
To validate the automated method, DNA for NGS library con-
struction was extracted from selected bacterial isolates with
different 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’s
instructions [9]. Before shearing, the extracted DNA was
analyzed using an Agilent 2200 TapeStation system with the
Genomic DNA ScreenTape assay for integrity of high
molecular weight DNA [7,10,11]. Fragmented DNA was quanti-
fied with the method described by Jeannotte et al. [7] using an
Agilent 2200 TapeStation system as the 96-well plate
high-throughput workflow for quantitation and sizing of gDNA
samples for library construction [9].
Table 1. Library Construction Metrics for Libraries Prepared from Different
Bacteria
Bacterium
Gram
reaction
Approximate
genome size
(MB)
GC
content
(%)
Average
library
size (bp)
Final
library
yield (ng)
Lactococcus Positive 2 35 430 679
Listeria Positive 2 38 307 949
Vibrio Negative 5 (two
chromosomes)
41 304 198
Escherichia Negative 5 51 347 361
Salmonella Negative 5 52 299 287
DNA was sheared in batches of 96 samples using microtubes
with the Covaris E220 Focused Ultrasonicator [12]. The frag-
mented DNA size was determined with the Agilent 2100
Bioanalyzer system and High Sensitivity DNA Kit, to confirm a
normal size distribution around a ~300 bp peak.
The input into library construction with the KAPA HTP Library
Preparation Kit (KK8234, Figure 2) was normalized to 1–5 µg
for all samples [13]. The standard KAPA protocol with
dual-SPRI size selection after adapter-ligation was used to
achieve libraries with a fragment distribution in the range of
250–450 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 High
Sensitivity DNA Kit [14,15]. Libraries were quantified with the
qPCR-based KAPA Library Quantification Kit (KK4824) prior to
normalization and pooling for sequencing [16] at BGI@UCD
(Sacramento, CA) on the Illumina HiSeq 2000.
Input DNA
Fragmentation
Adaptor ligation
Library amplification
End repair
A-tailing
Size selection
Figure 2. Detailed KAPA HTP Library Preparation protocol. The input into
library construction is fragmented DNA or cDNA. Each enzymatic
reaction is followed by a SPRI-bead cleanup (1.7X after End
Repair; 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 associated
with the physical transfer of material between enzymatic reac-
tions. This results in higher yields of adapter-ligated libraries, and
reduces the number of amplification cycles required to generate
sufficient material for Library QC and sequencing.

4
The entire library construction workflow, from fragmentation
to the post-amplification cleanup, can be completed in as little
as 9 hours. The validated, 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 avail-
able to provide the end user with the option of preparing
libraries without size selection, employing a SPRI-bead based
size selection, or performing off-deck size selection. The
second protocol is used for library amplification setup, and the
third for the post-amplification cleanup. This modular design
provides the end-user with the flexibility to complete library
construction over two days, or implement physical separation
of pre- and post-PCR procedures. The VWorks Forms are sup-
plemented by a Microsoft Excel workbook (Figure 4D) to facili-
tate reagent preparation and instrument setup, and can be
used to keep a complete record of each experiment.
Consumables used on the NGS Workstation were selected for
optimal performance. Labware movements and pipetting para-
meters for the KAPA HTP Library Preparation protocols were
carefully optimized to minimize reagent and consumable
waste and ensure equal or better library yields and quality
compared to manual preparation methods.
Figure 3. The Agilent NGS Workstation. This instrument comprises the
highly reliable Agilent Bravo Automated Liquid Handling Platform,
Agilent Bravo accessories for heating, cooling, shaking, and mag-
netic 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.

5
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) Library
Amplification Setup Form, C) Post-amplification Cleanup Form, D) Microsoft Excel workbook. Each run is configured by selecting the number of plate
sample 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 a
record of each experiment. The VWorks software has many additional, convenient features, including various ways of tracking the progress during a
run, and intuitive manual control of over instrument components to facilitate error recovery.
A B
C D

6
Results and Discussion
Prior to shearing, the extracted DNA was analyzed using the
Agilent 2200 TapeStation system with the Genomic DNA
ScreenTape assay (Figure 5). A typical electropherogram for
fragmented bacterial DNA used for library construction is
given 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
different apparent library fragment sizes, but all of these
libraries 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 of
adapter-ligated library was higher for Listeria than for the
other bacteria, the number of amplification cycles could have
been reduced. The enteric pathogens also produced similar
sized libraries that produced excellent sequence results.
Library construction metrics (average library size and final
library yields) for libraries prepared from different bacteria are
summarized in Table 1.
Figure 5. Typical electropherogram generated with the Agilent 2200
TapeStation system and Genomic DNA ScreenTape assay to
assess integrity of high molecular weight genomic DNA used for
library construction. Each line represents an individual Listeria
isolate.
600
500
400
300
[bp]
200
100
600
900
1,200
1,500
Sample intensity [FU]
2,000
2,500
3,000
4,000
7,000
15,000
48,500
0
Figure 6. A typical electropherogram (generated on the Agilent 2100 Bioanalyzer system and
High Sensitivity DNA Kit) of Covaris-sheared DNA used for library construction.
Fragmentation parameters were selected to produce a fragment size distribution with
a peak in the range of ~300 bp, which is ideal for 2 x 100 bp paired-end sequencing on
the Illumina HiSeq 2000.
250
[FU]
200
150
100
50
35 100 150 200 300 400 500 600 1,000 2,000 10,380 [bp]
D1
0

7
300
250
200
150
100
50
0
40 50 60 70 80 90 100 110 120 130 sec
300
250
200
150
100
50
0
40 50 60 70 80 90 100 110 120 130 sec
Ladder
200
150
100
50
0
40 50 60 70 80 90 100 110 120 sec
7,000
[bp]
2,000
1,000
600
500
400
300
200
150
100
35
Ladder
7,000
[bp]
2,000
1,000
600
500
400
300
200
150
100
35
Ladder
7,000
[bp]
2,000
1,000
600
500
400
300
200
150
100
35
150
200
100
50
0
40 50 60 70 80 90 100 110 120 130 sec
200
250
150
100
50
0
40 50 60 70 80 90 100 110 120 130 sec
Ladder
7,000
[bp]
2,000
1,000
600
500
400
300
200
150
100
35
Ladder
7,000
[bp]
2,000
1,000
600
500
400
300
200
150
100
35
Lactococcus, 35% GC
The average library size for
Lactococcus is 430 bp
Listeria, 38% GC
The average library size for
Listeria is 307 bp.
Vibrio, 41% GC
The average library size for
Vibrio is 304 bp.
Escherichia, 51% GC
The average library size for
Escherichia is 347 bp.
Salmonella, 52% GC
The average library size for
Salmonella is 299 bp
Figure 7. Representative electropherograms and virtual gel images (generated on the Agilent 2100 Bioanalyzer system with the High Sensitivity DNA Kit) of
bacterial libraries prepared for whole genome sequencing with the KAPA HTP Library Preparation Kit on the Agilent NGS Workstation. The average
library size for each genus was as indicated. Peaks at 35 and 10381 bp are internal standards used for alignment and quantitation determination with
the Agilent 2100 Bioanalyzer system.

www.agilent.com/chem
Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.
Information, descriptions, and specifications in this publication are subject to change
without notice.
© Agilent Technologies, Inc., 2014
Printed in the USA
May 19, 2014
5991-4296EN
Conclusion
The KAPA HTP Library Preparation Kit and Agilent NGS
Workstation provide a robust, high-throughput automation
solution for the construction of high-quality libraries for
microbial whole genome sequencing on the Illumina plat-
form. This combined solution produced a workflow that is
suitable for the construction of high-quality libraries for
microbial whole genome sequencing in a production scale
sequencing project 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, and
Kendra Liu from the laboratory of Dr. Bart Weimer at
University of California, Davis.
References
1. S. Fisher, et al., Genome Biology 12, R1 (2011).
2. M.A. Quail, et al., Nature Methods 9, 10–11 (2012).
3. S.O. Oyola, et al., BMC Genomics 13, 1 (2012).
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
Using Agilent DNA Extraction Kit (p/n 200600).
7. R. Jeannotte, et al. Agilent Technologies Application
Note (5991-4003EN): High-Throughput Analysis of
Foodborne Bacterial Genomic DNA Using Agilent 2200
TapeStation and Genomic DNA ScreenTape System.
8. KAPA Express Extract Kits: http://www.kapabiosys-
tems.com/products/name/kapa-express-extract-kits
9. Qiagen QIAamp DNA Mini Kit:
http://www.qiagen.com/products/catalog/sample-
technologies/dna-sample-technologies/genomic-
dna/qiaamp-dna-mini-kit
10. Agilent 2200 TapeStation User Manual, Agilent
Technologies (p/n G2964-90001).
11. Agilent Genomic DNA ScreenTape System Quick Guide,
Agilent Technologies (p/n G2964-90040 rev.B).
12. Covaris E220 Focused-ultrasonicators: http://covaris-
inc.com/products/afa-ultrasonication/e-series/
13. KAPA High Throughput Library Preparation Kit with
SPRI solution and Standard PCR Library
Amplification/Illumina series (96 libraries):
http://www.kapabiosystems.com/products/name/kap
a-library-preparation-kits
14. Agilent 2100 Bioanalyzer User Manual, Agilent
Technologies (p/n G2946-90003).
15. Agilent High Sensitivity DNA Kit Guide (Rev. B), Agilent
Technologies (p/n G2938-90321).
16. KAPA Library Quantification Kit - Illumina/Universal:
http://www.kapabiosystems.com/products/name/kap
a-library-quant-kits
For More Information
These data represent typical results. For more information
on our products and services, visit our Web site at
www.agilent.com/chem.