Large DNA Fragment Analysis: Constant- vs Pulsed-Field Electrophoresis Technologies
App Note / Case Study
Last Updated: October 11, 2023
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Published: September 8, 2023
Credit : Istock
Measuring the size and integrity of fragment DNA is an essential quality control step in next-generation sequencing (NGS) workflows. Hence, it is critical to choose the most appropriate electrophoresis technology to obtain accurate and reliable results.
Selecting the appropriate technology for analyzing HMW samples and large DNA smears is important to avoid problems such as DNA stacking or poor sizing.
This application note compares the use of pulsed- and constant-field technologies to support researchers in choosing the most suitable automated instrument for their sequencing workflow.
Download this application note to learn more about:
- Choosing the most suitable automated electrophoresis system for your needs
- Resolution differences between constant- and pulsed-field separation
- Quality control of HMW or sheared genomic DNA in NGS library preparation
Application Note
Genomics
Abstract
In next-generation sequencing (NGS) workflows, knowing the size and integrity
of DNA is important to determine sample input quality and measure the
success of library preparation. Electrophoresis is a common technique used
in genomic analysis labs to evaluate sample size and quality. Choosing the
appropriate electrophoresis technology is critical to achieving successful
downstream results. Constant- and pulsed-field electrophoresis technologies
are both designed to separate DNA based on size. Constant-field technology
is traditionally thought to be best suited for separation of DNA smears that are
less than 20 kb, based on previous studies.¹ Beyond 20 kb, DNA smears that are
analyzed using constant-field technology may be impacted by the compression
of samples, which occurs due to the stacking of DNA within the gel. Pulsed-field
technology is capable of separating DNA smears with no known compression
effects, as seen in constant-field technology. Agilent offers two capillary
electrophoresis instruments that use these different technologies: the Agilent
Fragment Analyzer systems use constant-field technology, while the Agilent
Femto Pulse system uses both pulsed- and constant-field technologies. The
data presented in this application note indicates that while both the Fragment
Analyzer and Femto Pulse can be utilized for analysis, the Femto Pulse provides
more accurate sizing for high molecular weight (HMW) DNA, genomic DNA
(gDNA) and sheared samples, within the range of 10 to 20 kb and above.
Comparison of Constant- and PulsedField Electrophoresis Technologies
for Analysis of High Molecular Weight
and Large DNA Fragments
Author
Timothy Butler
Agilent Technologies, Inc.
2
Introduction
Quality control (QC) is a crucial component of NGS workflows
as it provides valuable information about the size and quality
of the input sample, the intermediate steps, and the final
library. Knowing the size and quality can help users to make
informed decisions on whether their sample is suitable for
further downstream processes. QC steps are often performed
using electrophoresis to separate the sample based on size.
There are many types of electrophoresis technologies, so
choosing the most appropriate method for QC is important.
QC throughout NGS library preparation can be achieved using
both constant-field technologies like the Fragment Analyzer
systems, and pulsed-field technologies such as the Femto
Pulse system. This application note provides guidance on the
appropriate technology to use when analyzing HMW samples
and large DNA smears.
Constant-field electrophoresis technology relies on the
application of a constant current of electricity running
through a gel to separate DNA. This form of electrophoresis is
commonly used for smaller DNA fragments and smears less
than 20 kb in size.¹ An inherent problem with constant-field
electrophoresis is a phenomenon known as compression,
which can happen with large DNA samples. Compression
results from DNA stacking on top of itself as it moves through
the gel gradient. Larger DNA—usually greater than 20 kb in
size—is not able to be successfully separated and continues
to move as one mass.1
This lack of separation may cause
misrepresentation of the DNA smear size.
Pulsed-field electrophoresis technology is similar to constantfield electrophoresis but applies an oscillating current to the
sample during electrophoretic separation. The alternating
current of pulsed-field electrophoresis decreases the
compression that is seen with constant-field electrophoresis,
allowing for more effective separation of larger DNA fragments
and smears. The fluctuating current makes pulsed-field
technology ideal for QC analysis throughout a long-read
sequencing workflow. The lack of compression will cause DNA
to separate true to size, providing a clearer picture of a smear’s
distribution and more accurate sizing of large DNA samples.
The Fragment Analyzer and Femto Pulse systems offer
various kits that are suitable for both gDNA analysis and NGS
workflow QC. In this application note, the Fragment Analyzer
uses the Agilent Genomic DNA 50 kb kit to size samples
ranging from 75 to 60,000 bp, while the Femto Pulse employs
the Agilent Genomic DNA 165 kb kit and pulsed-field method
to size samples between 1.3 and 165 kb. The purpose of this
study was to compare the effectiveness of constant- and
pulsed-field technology for sizing DNA smear samples within
a specific range that both kits share. Specifically, the sizing
range tested was between 10 and 20 kb, which is similar to
the insert sample range of the PacBio HiFi SMRTbell library,
and the shearing range available through the Covaris g-TUBE
kit. Initially, the input gDNA was analyzed to confirm sample
size and integrity before shearing. Later, QC checks were
conducted on the sheared samples to ensure they met the
desired size for library preparation. This application note
indicates that while the Fragment Analyzer is affected by
compression in the 10 to 20 kb size range for smear samples,
the Femto Pulse is not. These findings can help researchers
choose the most suitable automated electrophoresis
instrument for their workflows.
Experimental
Genomic DNA input analysis with constant- and pulsedfield electrophoresis
A commercially available human gDNA sample (Promega,
part number G3041) was analyzed to ensure that it met
the minimum size requirement of 50 kb for shearing, as
recommended by the PacBio HiFi library protocol.²
The Agilent 5200 Fragment Analyzer system with the Agilent
Genomic DNA 50 kb kit (part number DNF-467-0500) was
used for constant-field analysis. The Agilent Femto Pulse
system with the Agilent Genomic DNA 165 kb kit (part
number FP-1002-0275) was used for pulsed-field analysis.
Sheared DNA sample analysis with constant- and pulsedfield electrophoresis
The gDNA was sheared using a g-TUBE (Covaris, part number
010145) to three different sizes. For each sample, 4 µg of
gDNA was added to a g-TUBE for shearing. Samples A and C
were prepared following the manufacturer’s instructions for
shearing to sizes of 10 and 20 kb respectively. Sample B was
prepared in the same manner but was centrifuged at 6,000
rpm to achieve a size of approximately 15 kb. Sheared gDNA
samples were analyzed on the Fragment Analyzer using the
Genomic DNA 50 kb kit and the Agilent HS Large Fragment 50
kb kit (part number DNF-464-0500). The same sheared gDNA
samples were run on the Femto Pulse using the Genomic
DNA 165 kb kit.
Data Analysis
Agilent ProSize data analysis software was used to assess
the quality of each sample that was analyzed on the Fragment Analyzer and Femto Pulse. Using smear analysis, each
sample was analyzed with a sizing range that incorporated
the whole smear.
3
Results
Resolution differences between
constant- and pulsed-field separation
To compare constant- and pulsed-field electrophoresis, it is
important to consider the separation resolution in the region
of interest. To demonstrate the differences in resolution, the
slope of the calibration curves for the Fragment Analyzer
Genomic DNA 50 kb kit and the Femto Pulse Genomic DNA
165 kb kit ladders were compared (Figure 1). To apply a
size to each sample, ProSize software plots time (X-axis)
against size (Y-axis) based on the ladder well. Electrophoresis
resolution is determined by the length of time required to
separate two fragments and the width of the peaks.³ A longer
separation time between two fragments leads to greater
Figure 1. The calibration curves of the ladders for the A) Agilent Fragment Analyzer system and the B) Agilent Femto Pulse system are shown. The blue dotted
lines on the curves correspond to the 10 and 48 kb ladder peaks for the C) Agilent Genomic DNA 50 kb kit ladder, analyzed on the Fragment Analyzer, and D) the 10
and 50 kb peaks of the Agilent Genomic DNA 165 kb kit ladder, on the Femto Pulse. The slope of the curves between the dotted lines demonstrate the resolution
capabilities of both instruments, with the Femto Pulse having a more gradual slope and thus higher resolution.
B. Fragment Analyzer Ladder
10 kb
48 kb
A. Fragment Analyzer Calibration Curve
10 kb
48 kb
D. Femto Pulse Ladder
10 kb
50 kb
C. Femto Pulse Calibration Curve
10 kb
50 kb
resolution and a more gradual slope in the calibration curve of
a ladder. A more gradual slope in the calibration curve allows
for more accurate sizing because the fragments are more
clearly distinguished from one another. To demonstrate this,
lines indicating the 10 kb ladder fragments and the 48 or 50
kb ladder fragments for the Fragment Analyzer and Femto
Pulse respectively are shown on the size calibration curve in
Figure 1. Within this range, the ladder slope for the Fragment
Analyzer (Figure 1A) is steeper than that for the Femto Pulse
(Figure 1C). The sharp increase in slope for the Fragment
Analyzer from 10 to 48 kb demonstrates the lower resolution
of constant-field electrophoresis for sizes greater than 10 kb.
In contrast, the Femto Pulse has a more gradual slope over a
longer period, thus leading to more accurate sizing of samples
from 10 to 50 kb.
4
Figure 2. Electropherograms of the same human genomic DNA sample
analyzed on the A) Agilent Fragment Analyzer system using the Agilent
Genomic DNA 50 kb kit, and B) Agilent Femto Pulse system using the Agilent
Genomic DNA 165 kb kit.
A
B
Quality control of HMW genomic DNA
QC of the input gDNA is commonly the first step in long-read
sequencing library preparation, such as in PacBio’s workflow.4
QC of gDNA can be performed with either constant- or
pulsed-field electrophoresis. To display this, the same
sample was run on the Fragment Analyzer and the Femto
Pulse. The Fragment Analyzer Genomic DNA 50 kb kit is
designed to size DNA fragments within a range of 75 bp to
60 kb based on the ladder. When using smear analysis in
the ProSize software, sizes larger than the ladder range can
be assessed by extrapolation of the ladder calibration curve,
enabling some analysis of HMW gDNA samples. The gDNA
electropherogram obtained from the Fragment Analyzer is
depicted in Figure 2A with a reported smear size of 64 kb.
Pulsed-field technology, which is utilized by the Femto Pulse
system using the gDNA 165 kb kit, has a sizing range of
1.3 to 165 kb. Samples larger than the gDNA 165 kb Ladder
can be extrapolated beyond the ladder to allow for higher
sizing when using the smear analysis function in the ProSize
software. Figure 2B displays the electropherogram for the
human gDNA analyzed on the Femto Pulse system, with a
total smear size of 215 kb.
The smear sizes between the two instruments were
measured with a 151 kb difference. Figures 2A and 2B appear
visually similar, but the Fragment Analyzer gDNA peak is
located close to its largest ladder size on the X-axis at 48 kb,
while the Femto Pulse peak is close to its largest ladder size
on the X-axis at 165 kb. This shows that the peak locations
are distinct from each other when comparing the two
technologies because of the difference in scale on the X-axis.
Based on the example presented, pulsed-field electrophoresis
is the preferred method for analyzing HMW gDNA due to its
ability to provide more accurate sizing.
Quality control of sheared genomic DNA
Following assessment of the initial gDNA sample, many NGS
protocols recommend shearing the sample to a size appropriate for the sequencing platform. For example, the PacBio HiFi
SMRTbell sequencing library preparation protocol requires
samples to be sheared to a target size distribution of 15 to
18 kb. In this study, Covaris g-TUBEs were utilized to prepare
samples of various sizes. All three samples (A, B, and C) were
analyzed on the Fragment Analyzer and Femto Pulse (Figure 3).
The data in Figure 3 demonstrates the comparison in sizing
capabilities of the two technologies for all three samples.
Sample A, with an expected size of approximately 10 kb,
showed little change in average size between constant- and
pulsed-field analysis. Sample B, expected to be approximately
15 kb in size, shows a sizing discrepancy between
constant- and pulsed-field electrophoresis. The size
reported with constant-field analysis for sample B was
approximately 20 kb. The pulsed-field method sized sample
B at approximately 15 kb (Figure 3). The variation between the
technologies grows more in sample C, which had an expected
size of 20 kb: the constant-field analysis sized the sample at
approximately 35 kb, but pulsed-field analysis sized sample C
at approximately 21 kb, on average.
The difference in sizing between constant- and pulsed-field
technologies becomes apparent with sample B and intensifies
as the shear size of sample C is reached. The bar chart in
Figure 3C illustrates this difference as sample shear size
increases. The observed difference seen is evidence
of compression acting on the constant-field sample.
Compression is most pronounced in sample C but, presented
here, compression begins between approximately 10 and
15 kb smear sizes. Thus, when a sample size is expected
to be above 10 kb, it is recommended that pulsed-field
electrophoresis technology such as the Femto Pulse is used
to achieve the most accurate sizing and integrity analysis of
the samples. To further confirm these results, all samples
were also run on the Fragment Analyzer with the HS Large
Fragment kit. The results shown on the HS Large Fragment
kit were consistent with the results shown from the
Fragment Analyzer Genomic DNA 50 kb kit (data not shown).
5
Figure 3. Electropherograms representative of sheared DNA smear analysis with sample A (black), expected size approximately 10 kb; B (blue), expected size
approximately 15 kb; and C (red), expected size approximately 20 kb, measured on the A) Agilent Fragment Analyzer system with Agilent Genomic DNA 50 kb
kit, and B) Agilent Femto Pulse system with Agilent Genomic DNA 165 kb kit. C) Bar chart comparing the smear size of each sample. D) Table listing the average
smear sizes for samples after shearing, measured on the Fragment Analyzer system using the Genomic DNA 50 kb kit, and the Femto Pulse using the Genomic
DNA 165 kb kit (n = 3).
Sample A Sample B Sample C
Genomic DNA
50 kb kit
Genomic DNA
165 kb kit
Genomic DNA
50 kb kit
Genomic DNA
165 kb kit
Genomic DNA
50 kb kit
Genomic DNA
165 kb kit
Average Size (bp) 13,028 11,649 20,398 15,089 35,142 21,466
%CV 3% 3% 6% 4% 1% 0%
A C
B
D
Size (bp)
38000
36000
34000
32000
30000
28000
26000
24000
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Fragment Analyzer gDNA 50 kb kit Femto Pulse gDNA 165 kb kit
Sample A Sample B Sample C
www.agilent.com/genomics/automated-electrophoresis
For Research Use Only. Nor for use in diagnostic procedures.
PR7001-0900
This information is subject to change without notice.
© Agilent Technologies, Inc. 2023
Published in the USA, June 01, 2023
5994-6103EN
References
1. Birren, B.; Lai, E. Pulsed field gel electrophoresis: A
practical guide. Acad. Pr. 1993
2. Procedure & Checklist - Preparing HiFi SMRTbell® Libraries
using the SMRTbell Express Template Prep Kit 2.0., Pacific
Biosciences of California Procedure & Checklist. Product
Number 101-853-100 Version 05, 2021.
3. Highly Resolved Separation of DNA Fragments on
the Agilent 5200 fragment Analyzer System, Agilent
Technologies application note, product number 5994-
0517EN, 2019.
4. Fast Accurate DNA Sizing with the Agilent Femto Pulse
System for HIFI WGS, Pacific Biosciences of California
Product Note. product number 102-326-561 Rev01, 2023
Conclusion
This application note demonstrates the abilities of constantand pulsed-field electrophoresis used by Agilent automated
electrophoresis systems to size input gDNA and sheared DNA
samples in the 10 to 20 kb size range. The results shown
here also demonstrate a phenomenon in constant-field
electrophoresis called sample compression. Compression
causes misrepresentation of the actual size of large DNA
smears. The data in this application note showed that as
the size of the samples increased, the difference between
the reported size of the constant- and pulsed-field data
increased. The data also points to compression beginning
between the sizes of approximately 10 and 15 kb. Constantfield technology, like the Agilent Fragment Analyzer, provides
accurate QC analysis for DNA smears smaller than 10 kb in
size, but can still be used for samples up to 60 kb. Pulsedfield technology, like the Agilent Femto Pulse system, is
preferred for QC of large DNA greater than 10 kb as it gives
better resolution and more accurate size determination.
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