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Improving Quality of DNA Fragments for Next Generation
Sequencing with Agilent 2200 Tapestation
Bao K. Luu1 Nguyet Kong1,2
Whitney Ng1,2
Lenore Kelly3
Bart Weimer1,2
1. School of Veterinary Medicine, U.C. Davis, Davis, CA
2. 100K Pathogenic Genome Project
3. Agilent Technologies, Inc., Santa Clara, CA
References Presenter’s Information
Contact Information
Workow Needs
Materials & Methods
Results and Discussions
High ResolutionEfcient
1. 2. 3. 4.
Single molecule real-time (SMRT) DNA sequencing technology system
can improve genome assembly by providing extra-long read lengths, ef-
fectively reducing the number of contigs.
The system’s application requires high-quality double stranded genomic
DNA (gDNA) of ~10 kb in size, so methods of DNA quantication and
sizing are critical.
Quality control (Q.C) of PacBio libraries with 2100 Bioanalyzer System
is currently considered gold standard, but its limited resolution of 12 kb
and 12 samples per run are not ideal for high throughput formats.
Campylobacter jejuni
• 30% GC
• Gram negative
• 2 MB
Listeria monocytogenes
• 30% GC
• Gram negative
• 2 MB
Vibrio uvials
• 30% GC
• Gram negative
• 2 MB
Salmonella enterica
serovar Enteritidis
• 30% GC
• Gram negative
• 2 MB
• High molecular weight gDNAs from microbes listed
above were extracted[3] and cleaned up with a Qiagen
QIAamp DNA Mini kit (51306).
• Extracted gDNAs were analyzed by the 2200 TapeSta-
tion System prior to shearing.
• gDNAs were sheared using Covaris g-TUBE devices[2].
• Size and quantity of fragmented gDNAs were deter-
mined with 2200 TapeStation System using the Genom-
ic DNA ScreenTape assay to conrm the normal size
of ~10 kb[4].
• PacBio libraries were constructed from fragment-
ed gDNAs using the PacBio SMRTbell 10 kb Library
Preparation kit.
Proper sizings of nal libraries were conrmed with
both the 2100 Bioanalyzer and 2200 TapeStation sys-
Libraries were quantied with a Life Technologies Qu-
bit 2.0 Fluorometer and a dsDNA HS assay (Q32854).
• Libraries were submitted to sequencing facility.
Q.C with Agilent 2200 Tapestation
Extracted High Molecular (HMW) gDNA
• The 2100 Bioanalyzer System could not assess extracted gDNAs (>50 kb) because of its limited resolution (12 kb).
• Quality control was thus assessed with the 2200 TapeStation System, 260/280 nm and 260/230 nm ratios (See Figure 1).
Figure 1. Electropherogram (A) and virtual gel (B)
of high molecular gDNA from an Agilent 2200
TapeStation using the Genomic DNA ScreenTape
Sheared HMW gDNA
• Fragment sizes were determined using the 2200 TapeStation System due to the high resolution (60 kb) of the Genomic
DNA ScreenTape Assay.
• The assay analyzed at the rate of 1-2 minutes per sample with the capacity of 96 samples per run.
• The sizes of many DNA fragments are within the upper range of 2100 Bioanalyzer System (See Figure 2).
Figure 2. Electropherogram (A) and virtual gel (B)
of average sheared gDNAs from
(16 kb),
(12 kb),
(14 kb), and
(20 kb).
Final Library Constructs
• 2200 TapeStation System was better at resolving the libraries without any interference (see Figure 3).
When conrming the library’s sizes with 2100 Bioanalyzer System, samples overlapped with the upper marker (see Figure 4).
Figure 3. Electropherogram (A) and virtual gel (B)
of average DNA libraries from
(12 kb),
(14 kb), and
(20 kb). All DNA libraries were prepared with Pac-
bio SMRTbell 10kb Library Preparation Kit.
Figure 4. Comparison of accuracy in length
determination of average DNA libraries be-
tween TapeStation and Bioanalyzer systems.
Bioanalyzer system is less accurate for DNA
libraries with lengths near the 12 kb upper
Bart C. Weimer, PhD. -
Nguyet Kong -
U.C. Davis (VM: PHR) VetMed 3B - Room 4016
1089 Veterinary Medicine Drive
Davis, Ca 95616
(530) 752 - 6426
1. Application Note‘s DOI - 10.13140/RG.2.1.4339.4644
2. Covaris User Manual g-TUBE (p/n 010154).
3. N. Kong,
et al
. “Production and Analysis of High Molecular Weight Genomic DNA
for NGS Pipeline Using Agilent DNA Extraction Kit”.
Agilent Technologies,
4. R. Jeannotte,
et al
. “High-Throughput Analysis of Foodborne Bacterial Genomic
DNA ScreenTape System.”
Agilent Techonologies
, 2014.
Bao K. Luu | (408) 300 - 2251
B.S. Neurobiology, Physiology and Behavioral Science
University of California, Davis
Undergraduate Class of 2018
• The classical approach to analyze PacBio 10 kb libraries has high degrees of inaccuracy, as libraries that are greater than 10 kb can merge into
the upper marker.
Accurate quantication and sizing of libraries are important for primer annealing and polymerase binding, so the 2200 TapeStation System is a
better choice with its high resolution.
• The versatility and convenience of using only the 2200 TapeStation System for quality control of all steps in PacBio library construction are
advantageous in a high-throughput sequencing pipeline.
DNA Extraction
Shear gDNA
Library Making
ResearchGate has not been able to resolve any citations for this publication.
Technical Report
Full-text available
The Agilent DNA Extraction Kit (p/n 200600) was compared to standard methods such as beadbeating and enzyme treatment for preparation of genomic DNA from the prokaryote Listeria monocytogenes. Using this extraction kit, with modifications, to lyse the bacteria and isolate high molecular weight DNA reproducibly yielded high quality DNA suitable for further applications such as polymerase chain reactions to produce amplicons, or for next-generation DNA sequencing. The quality of the high molecular weight DNA, and the comparison of extraction methods, was shown on the Agilent 2200 TapeStation with the Agilent Genomic DNA ScreenTape (p/n 5067-5365) and Agilent Genomic DNA Reagents (p/n 5067-5366). 2
Technical Report
Full-text available
The initial step in Next Generation Sequencing is to construct a library from genomic DNA. To gain the optimum result, extracted DNA must be of high molecular weight with limited degradation. High-throughput sequencing projects, such as the 100K Pathogen Genome Project, require methods to rapidly assess the quantity and quality of genomic DNA extracts. In this study, assessment of the applicability of the Agilent 2200 TapeStation was done using genomic DNA from nine foodborne pathogens using several accepted high-throughput methods. The Agilent 2200 TapeStation System with Genomic DNA ScreenTape and Genomic DNA Reagents was easy to use with minimal manual intervention. An important advantage of the 2200 TapeStation over other high-throughput methods was that high molecular weight genomic DNA quality and quantity can be quantified apart from lower molecular weight size ranges, providing a distinct advantage in the library construction pipeline and over other methods available for this important step in the Next Generation Sequencing process.
Application Note's DOI
Application Note's DOI -10.13140/RG.2.1.4339.4644