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

Figure 1: 100K Pathogen Genome Project sample preparation workflow for multiplexed, short-read Illumina sequencing Figure 3: 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. The "with-bead" protocol uses a single aliquot of SPRI beads for all cleanups prior to library amplification, producing higher yields of adapter-ligated libraries, and reduces the number of amplification cycles to generate sufficient material for Library QC and sequencing. Figure 2: Representative electropherograms of Listeria (generated on the Agilent 2100 Bioanalyzer system and Agilent 2200 TapeStation system) of bacterial libraries prepared for whole genome sequencing with the KAPA HTP Library Preparation Kit. 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. ABSTRACT A 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

DNA
extraction
QC: DNA
quality and
quantity
Shear DNA QC:
fragmentation
Library
construction
Library
normalization,
pooling &
sequencing
Input DNA
Fragmentation
End repair
A-tailing
Adaptor
ligation
Size selection
Library
amplification
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
DOI 10.6084/M9.FIGSHARE.1386854
Megan Lew 1, Nguyet Kong1, Kao Thao 1, Carol Huang 1, MarykeAppel 2,, Stephen Lappin 3, Lisa Knapp3, Lenore Kelly3, and Bart C. Weimer 1
1School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; 2Kappa Biosystems Inc., Wilmington, MA, USA; 3Agilent Technologies Inc., Santa Clara, CA, USA
Figure 1:
100K Pathogen Genome Project sample preparation workflow for multiplexed, short-read Illumina sequencing
Figure 3:
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. The “with-bead” protocol uses a single aliquot of SPRI beads for all cleanups prior to library
amplification, producing higher yields of adapter-ligated libraries, and reduces the number of amplification cycles to generate sufficient
material for Library QC and sequencing.
Figure 2:
Representative electropherograms of Listeria (generated on the Agilent 2100 Bioanalyzer system and Agilent 2200 TapeStation
system) of bacterial libraries prepared for whole genome sequencing with the KAPA HTP Library Preparation Kit. 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.
ABSTRACT
A 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.
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.
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, 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
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, low-bias NGS library
amplification [2,3,4,5].
METHODS
DNA Extraction
The DNA for the NGS library construction was extracted from selected
bacterial isolates with different GC content. Organisms were lysed
using the KAPA Express Extract Kit, after which the DNA was purified
with a Qiagen QIAmp DNA Mini Kit. 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].
DNA Fragmentation
The 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 the
High Sensitivity DNA Kit, to confirm a normal size distribution around
a 300 bp peak.
Library Construction
The samples were normalized to 1-5ug for all samples [13]. 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 prior to normalization and
pooling for sequencing [16] on the Illumina HiSeq 2000.
CONCLUSION
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.
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
ACKNOWLEDGEMENTS
I would like to acknowledge and appreciate all the help and
support received from everyone in Dr. Bart Weimer’s Lab.
CONTACT INFORMATION
Bart C. Weimer, Ph D. (bcweimer@ucdavis.edu)
Nguyet Kong (nudao@ucdavis.edu)
Megan Lew (meglew@ucdavis.edu)
UC Davis (VM:PHR) VetMed3B 4016
1089 Veterinary Medicine Drive
Davis, Ca 95616
(530) 752-6426
Bacterium Gram
Reaction
Approx
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 41 304 198
Escherichia Negative 5 51 347 361
Salmonella Negative 5 52 299 287
Genomic DNA Fragmented DNA Final Library