Improving Quality of DNA Fragments for Next Generation Sequencing with Agilent 2200 Tapestation

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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
Background
Workow Needs
Materials & Methods
Results and Discussions
Conclusion
Automated
Consistent
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
System.
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
Campylobacter
(16 kb),
Listeria
(12 kb),
Vibrio
(14 kb), and
Salmo-
nella
(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
Campylobacter
(16
kb),
Listeria
(12 kb),
Vibrio
(14 kb), and
Salmonella
(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
marker.
Bart C. Weimer, PhD. - bcweimer@ucdavis.edu
Nguyet Kong - nugdao@ucdavis.edu
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,
2014.
4. R. Jeannotte,
et al
. “High-Throughput Analysis of Foodborne Bacterial Genomic
DNA ScreenTape System.”
Agilent Techonologies
, 2014.
Bao K. Luu
Baoluu@ucdavis.edu | (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
Sequencing