Production and Analysis of High Molecular Weight Genomic DNA for NGS Pipelines Using Agilent DNA Extraction Kit (p/n 200600)

Abstract

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

Full text

Production and Analysis of High
Molecular Weight Genomic DNA for
NGS Pipelines Using Agilent DNA
Extraction Kit (p/n 200600)
Authors
Nguyet Kong, Winnie Ng, Vivian Lee,
and Bart C. Weimer
University of California, Davis
Davis, CA 95616
Lenore Kelly
Agilent Technologies, Inc.
Santa Clara, CA 95051
Application Note
Abstract
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 reac-
tions 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
Introduction
This application note demonstrates the use of an Agilent DNA
extraction kit with microbial samples. The Agilent 2200
TapeStation system, using Listeria monocytogenes, was
employed for the assessment of genomic DNA (gDNA) sam-
ples sizing and quantification. High molecular weight gDNA
(HMgDNA) is required for many different assays. DNA for
polymerase chain reaction (PCR) was used with an Agilent
2100 Bioanalyzer system to assess PCR amplicons.
Production of HMgDNA is critical to the successful DNA
sequencing of microbial genomes. It is possible to sequence
the genome of many types of bacteria, but one of the limita-
tions is the ability to lyse the microbe and collect HMgDNA of
sufficient quality for use in next generation sequencing (NGS).
The 100K Pathogen Genome Project (http://100kgenome.
vetmed.ucdavis.edu/) prepares hundreds of DNA samples
each day for genome sequencing. As part of that effort, and to
use NGS as part of a food-borne outbreak investigation, a pro-
tocol that lyses bacterial cells to release HMgDNA ready for
sequencing with little to no previous experience is required.
L. monocytogenes, a gram positive, intracellular pathogen, is
a major public health concern in the United States due to its
high mortality rate and presence in the food supply. In the
USA, one in six people becomes ill from eating contaminated
food each year. L. monocytogenes accounted for 621 docu-
mented cases of food-borne illnesses in the USA during 2011,
with 22% leading to death [1]. We selected this organism
because of the difficulty of lysing gram-positive bacteria and
the need to have a fast and robust method that consistently
results in HMgDNA.
The Agilent DNA extraction kit (cat #200600) was used, with
some modifications, to lyse and extract HMgDNA from
L. monocytogenes. Following extraction, the DNA quality and
quantity was measured with the 2200 TapeStation system to
verify the HMgDNA before any NGS work. The extracted
gDNA can be used for polymerase chain reaction (PCR), and
the Agilent 2100 BioAnalyzer can be used for assessment of
PCR amplicons. PCR amplification was done as described by
the kit instructions (Black Diamond Genomics, Valrico, FL)
using the Listeria primer pair to verify the quality of DNA from
the isolation method.
Experimental
Bacteria
L. monocytogenes was cultured in Difco BHI Broth (Franklin
Lakes, New Jersey) at 37 °C aerobically for 12–16 hours
before using it to collect DNA. The amount of cells were nor-
malized to a 600 nm (optical density at 600 nm) = 0.2, which is
equal to ~1 x 108cells/mL. For this experiment, 1 mL of the
cultured broth was used to collect a cell pellet for genomic
DNA extraction after centrifugation at 20,000 xg for 5 minutes.
Genomic DNA extraction
Genomic DNA was extracted from cell pellets and split for use
with each of the testing methods. Each method was tested
using three replicates.
•Method 1: Commercial DNA extraction kit with silica spin
column [2,3]
•Method 2: Beadbeating with silica spin column [4,5]
•Method 3: Lysozyme and mutanolysin lysis and extraction
[6,7]
•Method 4: Agilent lysing buffer containing SDS with silica
spin column [8]
•Method 5: Agilent DNA extraction with modified protocol
[this study]

3
The Agilent kit was modified for extraction as follows [9]:
1. Suspend collected cell pellet in 1 mL of Solution 2 and
vortex until mixed throughly.
2. Add 1 µL of pronase. Incubate at 60 °C for 1 hour.
3. Place on ice for 10 minutes.
4. Add 500 µL of Solution 3 and invert to mix completely.
5. Place on ice for 5 minutes.
6. Centrifuge at 4,000 xg at 4 °C for 15 minutes and transfer
supernatant to a new 1.5-mL microcentrifuge tube.
7. Centrifuge at 20,000 xg at 4 °C for 5 minutes and transfer
supernatant to new 1.5-mL microcentrifuge tube.
8. Add 3 µL of RNase to the supernatant and invert to mix
completely.
9. Incubate at 37 °C for 15 minutes.
10. Split supernatant into two 1.5-mL microcentrifuge tubes
and add an equal volume of isopropanol to each tube.
11. Invert to mix completely.
12. Incubate on ice for 30–60 minutes.
13. Centrifuge DNA at 4,000 xg at 4 °C for 15 minutes.
14. Remove the supernatant (that is, isopropanol).
15. Fill tube containing the DNA pellet to rim with 70%
ethanol.
16. Mix completely, centrifuge at 20,000 xg for 5 minutes, and
discard ethanol.
17. Air dry and DNA and rehydrate DNA with 100 µL of water
and resuspend.
18. Combine both tubes into a single sample. This yields
200 µL DNA.
Genomic DNA analysis
After cell lysis and DNA extraction, the samples were placed
in a 96-well plate for the initial quality assessment using the
NanoDrop 1000 (NanoDrop Technologies) spectrophotometer.
Subsequently, the 2200 TapeStation with the Agilent Genomic
DNA ScreenTape (p/n 5067-5365) and Agilent Genomic DNA
Reagents (p/n 5067-5366) [10] were also used to evaluate the
size of the gDNA, quantification, and quality of the extracted
gDNA [11]. Following manufacturer guidelines, the first well
of the 96-well plate contained the 3 µL of the Genomic DNA
Ladder. Each well following the ladder contained 1 µL of the
extracted genomic sample with 10 µL of the Genomic DNA
Sample buffer, and the 96-well plate was vortexed on high
speed for a few seconds with a brief centrifugation at 500 xg.
Then, the plate was placed into the 2200 TapeStation. Each
extraction method was analyzed for size and quantity.
This protocol was tested with 200 different isolates of
L. monocytogenes to determine the consistency over
individual isolates.
Results and Discussion
The concentration, quality and molecular weight was mea-
sured for the gDNA against each lysis and extraction method
[12]. The 260 nm/280 nm and 260 nm/230 nm ratios were
used to quickly assess the contamination of the gDNA with
protein or organics, respectively. The ratio of ¡1.8 is accept-
able to proceed with additional DNA assessment (Figure 1).
Figure 1. Mean quantification data by NanoDrop including standard error
mean (SEM) bars for each extraction method.
2.5
2.0
A260/A280
A260/A230
1.5
Absorbance ratios
Sample NanoDrop reading
1.0
0.5
0
Method 1
Method 2
Method 3
Method 4
Method 5

4
Methods 2, 3, and 5 produced gDNA of sufficient quality to
proceed with additional assessment of HMgDNA with the
2200 TapeStation. The Genomic DNA ScreenTape used the
lower marker of the DNA ladder to quantify the samples in
each lane (Figure 2). Methods 2, 3, and 5, produced HMgDNA,
which appears as the intense largest band in each lane.
Using the electropheogram mode, each sample was mea-
sured to determine the total DNA size and quantity.
(Figure 3A–E). HMgDNA size ¡50 kb will be acceptable DNA
for NGS pipeline.
After demonstrating that the modified Agilent DNA extraction
kit lysed and extracted HMgDNA, an additional 200
L. monocytogenes samples were processed using the
modified protocol.
Figure 2. Each replicate of gDNA was examined using the TapeStation to determine degradation and
amount of HMgDNA produced from each method.
Method 1Ladder Method 2 Method 3 Method 4 Method 5
500
600
400
300
Sample intensity (FU)
200
100
0
bp
600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,000
48,500
Figure 3A. Electropherogram for extraction Method 1: Commercial DNA
extraction kit. In all figures, the internal standard is shown at
100 base pairs.
Figure 3B. Electropheogram for extraction Method 2: Beadbeating.
900
1,000
700
500
Sample intensity (FU)
300
200
100
400
600
800
bp
600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,000
48,500

5
Figure 3C. Electropheogram for extraction Method 3: Lysozyme and
mutanolysin. Figure 3D. Electropheogram for extraction Method 4: Lysis buffer
containing SDS.
Figure 3E. Electropheogram for extraction Method 5: Agilent DNA
Extraction Kit with modified protocol.
500
Sample intensity (FU)
300
200
100
400
600
bp
600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,000
48,500
500
Sample intensity (FU)
200
100
400
300
600
0
bp
600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,000
48,500
600
Sample intensity (FU)
300
200
100
500
400
0
bp
600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,000
48,500

6
A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 A3 B3C2 D2 E2 F2 G2 H2
Figure 6. Example image of DNA isolated from Listeria. This is a representative example from 200 different isolates using the
TapeStation to determine the gDNA size using extraction Method 5: modified Agilent DNA extraction.
Figure 4. NanoDrop readings of isolates with 99% passed (198/200). The
ratio of
¡
1.8 ratio is acceptable as pure DNA. Average
A260/A280 is 1.9
\
0.08 and average A260/A230 is 2.1
\
0.16.
Figure 5. Overlay of electrophoresis showing molecular weight for each
Listeria isolate sample. It illustrates the extraction efficiency
using Method 5: modified Agilent DNA extraction method.
4
3
2
1
0
A260/A280
Absorbance ratios
Sample NanoDrop readings
DNA quality ratio from 200 different Listeria isolates using
extraction Method 5: modified Agilent DNA extraction
A260/A230
600
Sample intensity (FU)
300
200
100
500
400
700
0
bp
600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,000
48,500

7
Conclusion
Genomic DNA from Listeria using Extraction Method 5 (as
shown in Figures 4–6) was also used to demonstrate that the
DNA was of sufficient quality to enable PCR and was large
enough to use for other applications, such as NGS.
Methods 2 and 3 were used as controls, since they are
accepted protocols; however, these methods often produce
lower molecular weight DNA. The modified protocol with the
Agilent DNA extraction kit consistently produced HMgDNA
similar to the classical lysing method containing lysozyme
and mutanolysin, but without the additional cost of enzymes
and in a simplified procedure that is amenable to automation
or use by less experienced operators.
Acknowledgment
We gratefully acknowledge the technical assistance provided
by Kao Thao, Kerry Le, Sum Leung, Sue Thao, Christina Kong,
Lucy Cai, Alan Truong and Alvin Leonardo.
Figure 7. Example image of QC amplicion to verify the gDNA isolated from Listeria using extraction Method 5 (modified Agilent extraction) produced DNA of
sufficient quality for PCR. This is a representative example from 200 different isolates using the Agilent 2100 Bioanalyzer with the DNA 1000 chip.
bp
1,500
1,000
700
500
400
300
200
150
100
50
15
L12345678910 11 12
Ladder Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12

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December 31, 2013
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