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Biol.J.Biotechnol.
200 J. Mol.
AsPac
Mol. Biol.2014
Biotechnol. Vol. 22 (3), 2014
Vol. 22 (3) : 200-208
Optimization of DNA shearing by sonication
Optimization of genomic DNA shearing by sonication
for next-generation sequencing library preparation
Le Jie Lee1,2 and Maha Abdullah1*
Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor Malaysia
2
Malaysia Genome Institute, National Institutes of Biotechnology Malaysia, Jalan Bangi, 43000 Kajang, Selangor Malaysia
1
Received 2nd October 2014/ Accepted 18th November 2014
Abstract. Next-generation sequencing (NGS) technologies, capable of sequencing genomic DNA and RNA at high throughput
with unprecedented speed, have revolutionized genomic research as well as clinical diagnosis. DNA fragmentation is a critical
step in library preparation in all NGS platforms, and determines the quality and diversity of the final library. DNA shearing by
acoustic sonication is one of the ways to randomly break DNA into small fragments, however many variables affect the outcome.
Here, we describe an optimized procedure to shear genomic DNA into fragments of 150 bp to 120 bp using a focused-ultrasonicator.
Parameters that were assessed included DNA quantity, the effect of repeat shearing, treatment time, peak incident power and
shearing reproducibility. This input of pure and optimum quality DNA samples is an essential starting point to the NGS system. We
identified peak incident power as being the key determining factor in obtaining small target fragments. By increasing the peak
incident power to 75W, a peak size within the 150 bp to 200 bp range was achievable, a result which was reproducible in multiple
samples. Repeat shearing and increased treatment time were less successful in producing optimally sized DNA fragments. The
proposed method may be used as a guide for NGS users involved in library construction, particularly when small fragment sizes
are required.
Keywords: DNA shearing, Next-generation sequencing, NGS library preparation, Sonication.
INTRODUCTION
The Human Genome Project instigated a revolution
in DNA sequencing methods and led a drive to overcome
old limitations. New technologies were able to process
hundreds of thousands to millions of DNA templates in
parallel, resulting in low cost and throughput on the
gigabase scale (Lander et al., 2001; McPherson et al., 2001;
Sachidanandam et al., 2001). Newer methods in
sequencing technology are referred to as next-generation
sequencing (NGS) as compared to conventional Sanger
sequencing (Mamanova et al., 2010). With the
high-throughput parallel NGS sequencing platforms,
complete sequencing of a whole human genome can be
accomplished in less than three days at a cost of USD $1000
(Anon, 2014).
The various available NGS technologies rely on
different strategies to combine template preparation,
sequencing, imaging, genome alignment and assembly
methods. NGS platforms provided by different service
providers may be based on different combinations of
sequencing chemistry such as Illumina (sequencing by
synthesis), Roche 454 (pyrosequencing), SOLiD
(sequencing by ligation), Ion Torrent (ion semiconduc-
tor) and Pacific Biosciences (single-molecule real-time
sequencing) (Liu et al., 2012; Quail et al., 2012a). The
preparation of a high quality sequencing library plays a
crucial role in NGS. The general workflow of NGS thus
starts with the random breaking of entire genomic DNA
into small fragments then ligating those small fragments
of genomic DNA to designated adapters for random read
during DNA sequencing (Zhang et al., 2011). Fragment
sizes are system dependent (Pushkarev et al., 2009). There
are three main ways to randomly shear DNA into short
fragments compatible for NGS: 1) physical fragmentations
(acoustic shearing, sonication, or hydrodynamic shear);
2) enzymatic methods (using restriction endonucleases,
non-specific nucleases, or transposases) and 3)
chemical fragmentation (using heat, divalent metal cation)
(Knierim et al., 2011). Some fragmentation protocols might
* Author for correspondence: Dr. Maha Abdullah,
Immunology Unit, Department of Pathology, Faculty of Medicine
and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang,
Malaysia.
Tel.: 603-89472375, Fax: 603-89412787,
Email: [email protected].
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (3), 2014
carry over chemicals or enzymes that will affect the read
qualities. Thus, comparison between libraries constructed by
sonication and enzymatic shearing was made, since the former
would have the advantage of avoiding carry-over; both were
found to be effective (Quail et al., 2012b). However, the
enzymatic method produces a greater number of artifact
insertions/deletions in raw sequence reads compared with
samples prepared by physical sonication (Knierim et al.,
2011).
Genomic DNA shearing by acoustic sonication is thus
one of the preferred methods for preparation of high quality,
random and size-appropriate NGS libraries. In this study,
we describe various parameters to consider during DNA
shearing and the optimization of a procedure for shearing
genomic DNA intro fragments in the range of 150 bp to
200 bp using a focused-ultrasonicator.
MATERIALS AND METHODS
DNA samples.
DNA samples were isolated from
peripheral blood mononuclear cells obtained from healthy
volunteers recruited for this study, which was approved
by the Institutional Medical Research Ethics Committee,
Universiti Putra Malaysia. Informed consents were obtained.
Isolation of mononuclear cells and DNA extraction.
Peripheral blood mononuclear cells were isolated
from whole blood samples using density gradient
centrifugation on the Ficoll-Paque Plus platform (GE
Healthcare, Sweden) according to the manufacturer’s
protocol. Isolated mononuclear cells were washed with
1× saline phosphate buffer and then suspended in 3 ml of
nucleic acid lysis buffer (10 mM Tris-HCl, 400 mM
NaCl and 2 mM Na2 EDTA, pH 8.2). The salting out
procedure for extracting genomic DNA described by
Miller et al. (1988) was performed after lysis of mononuclear
cells. Extracted DNA was dissolved in TE buffer (10 mM
Tris-HCL, 0.2 mM Na2 EDTA, pH 7.5).
Pre-fragmentation analysis of DNA quality. DNA quality
was determined by measuring absorbance at 260 nm (A260),
280 nm (A280) and 230 nm (A230) with a Nanodrop 1000
Spectrophotometer (Thermo Scientific, USA). The A260/280
and A260/230 ratio values were used to estimate DNA purity.
DNA integrity was confirmed by running an aliquot of each
DNA sample on a 2% agarose gel stained with ethidium
bromide.
DNA quantity was also determined using the Qubit
dsDNA assay kit (Life Technologies, USA) and analysis
using the Qubit Fluorometer (Invitrogen, USA).
Optimization of DNA shearing by sonication
201
DNA shearing parameters.
The Covaris M220
Focused-ultrasonicator (Covaris, USA) was used for DNA
shearing, set to produce DNA fragments with a target peak
size range of 150 bp to 200 bp, that which is required for
subsequent steps of SureSelect target enrichment (Agilent
Technologies, USA) library preparation. The parameters
provided in the user guide to obtain a target base pair peak
of 150 bp were as follows: 50W of peak incident power,
20% of duty factor, 200 cycles per burst, and 350 seconds of
treatment time at 20 oC. However, other parameters require
optimization to the individual lab setting and user. In this
study Peak incident powers of 50W, 70W and 75W were
tested, an extra 20 seconds was added to treatment time to
examine the shearing effect in an extended treatment time,
the effect of repeat shearing on sheared DNA was examined, and two different DNA quantities, 3.5 µg and 5.0 µg,
were prepared for shearing. The recovered DNA yield was
compared after the shearing, and the reproducibility of the
optimized method was tested using ten biological replicates.
Purification of fragmented DNA and post-fragmentation
DNA quality analysis. The library of sheared DNA was
purified using Agencourt AMPure XP beads (Beckman
Coulter, USA) according to the manufacturer’s instruction.
The purified DNA was eluted in 50 µl nuclease-free water
(Qiagen, USA).
The quality of DNA, including the distribution of
fragment length and peak height, was assessed using a
2100 Bioanalyzer (Agilent Technologies, USA). An Agilent
DNA 1000 chip and a High Sensitivity DNA chip (Agilent
Technologies, USA) were used to run the sheared DNA
library data, and the electropherogram was analyzed by
Agilent 2100 Expert Software version B.02.02.
RESULTS AND DISCUSSION
Pre-fragmentation DNA quality.
A260/280 and A260/230
ratios and concentrations of extracted DNA were
measured by both Nanodrop 1000 Spectrophotometer
and Qubit Fluorometer are shown in Table 1. The A260/280
ratio is used to indicate protein contamination of DNA;
generally, a ratio in the range of 1.8-2.0 indicates good
quality (Sambrook et al., 2001). The A260/230 ratio is used
as a secondary measure of DNA purity; absorption at 230
nm can be caused by contaminants other than protein
such as phenolate ions, thiocyanate, organic compounds or
polysaccharides. Expected A260/230 ratios in pure DNA are
commonly in the range of 2.0-2.2 (Turner et al., 2005).
Only samples producing values within these
ranges were selected to proceed with DNA shearing. It was
noticed that concentrations measured by the Qubit
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Optimization of DNA shearing by sonication
Table 1. Quality of DNA (A260/280 and A260/230 ratios and
concentrations
measured
by
Nanodrop
1000
Spectrophotometer and Qubit Fluorometer) from ten
genomic DNA samples extracted from mononuclear cells of
human blood.
Sample Ratio Ratio Concentration
number A260/280 A260/230 measured by
Nanodrop
(ng/µl)
1
2
3
4
5
6
7
8
9
10
1.85
1.80
1.84
1.88
1.84
1.85
1.85
1.86
1.82
1.84
2.29
2.28
2.18
2.20
2.25
1.97
2.28
2.24
2.15
2.11
596.5
458.1
310.0
224.7
387.5
208.6
1046.5
954.1
343.1
244.3
Concentration
measured by
Qubit (µg/ml)
137.3
314.7
148.0
130.0
133.0
121.3
330.7
513.7
185.0
108.7
Fluorometer were 1.5 to 4 times lower in all samples
compared to the concentrations measured using the
Nanodrop 1000 Spectrophotometer. Based on other
studies, it is common for spectrophotometry to overestimate
the concentration, giving values ranging from 2 up to 30
times greater than their actual concentration (Mee et al.,
2011; Sironen et al., 2011; Mathot et al., 2013). Although
spectrophotometry is the most common method used to
quantify nucleic acid, it can be unreliable and inaccurate
(Clasel, 1995). This is because DNA is not the only molecule
that can absorb UV light at 260 nm. Contaminants that will
affect the purity of DNA such as RNA are highly absorbant
at 260 nm, aromatic amino acids absorbing at 280 nm will
contribute to the final measurement at these wavelengths,
and the nucleotide guanidine gives an even higher value
for 260 nm absorbance (Manchester, 1996). So, the major
disadvantages of spectrophotometry are insensitivity
and the tendency to overestimate the DNA quantity. As
fluorometric technology utilizes specific probe dyes
that will only bind to double-stranded DNA, the
specificity and sensitivity are higher than the
conventional spectrophotometry method in which the
absorbance reading is affected by the background noise of
contaminants (Georgiou and Papapostolou, 2006; Shokere
et al., 2009; Singer et al., 1997). Thus, Qubit dsDNA
assay is suggested as a more accurate method to measure the
concentration of DNA for NGS library preparation.
Apart from the quality, the integrity of extracted DNA
is also important for DNA shearing. Figure 1 shows the
image of extracted DNA samples visualized by 2%
agarose gel electrophoresis to assess the degree of
degradation. Although extracted DNA is meant to be
fragmented for downstream construction of NGS library,
degraded or partially degraded DNA may produce smaller
fragments that will affect average distribution of the desired
target fragment length (Simbole et al., 2013).
Parameters affecting DNA fragmentation. The initial
quantity of DNA affects the outcome of shearing. The
average recovery of DNA after shearing of 3.5 µg DNA
was 70.2% (±7.3) while with 5.0 µg the average recovery
increased to 77% (±18.2). DNA recovery after
shearing however might be sample dependent. Therefore, it is
recommended that a higher starting amount of DNA is
used to increase the yield since the maximum DNA input
for Covaris M220 is 10 µg. However, too great a DNA input
will also result in incomplete shearing causing tailing of the
electropherogram where the majority of the DNA remains
in larger sizes.
Figure 1. Gel image of 10 genomic DNA samples
extracted from mononuclear cells of human blood (lanes
labelled 1 to 10) run on a 2% agarose gel. Single bands indicated that the
extracted genomic DNA samples were intact and not degraded.
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (3), 2014
The quality of DNA shearing is assessed as peak heights
on electropherogram from the Bioanalyzer DNA 1000
chip (Table 2). The average peak size of ten fragmented
specimens using the manufacturer’s recommended
parameters was 205.3 bp (±15.4). The peaks produced by
most of the samples were higher than the target of 150
bp to 200 bp and the electropherogram of the sheared
DNA samples showed a very broad distribution of
fragment sizes (Figure 2). Broad distribution of fragments
indicates that there were a lot of fragments present that were
smaller or larger than the target range. As seen in the overlaid
electropherograms in Figure 2, the majority of the samples
produced distribution of post-shearing fragment sizes that
skewed to the right, meaning that the average size of the
sheared DNA fragments was still larger than 150 bp to 200
bp. Indeed most of the sheared DNA samples had fragment
lengths above 200 bp, and so an optimization of parameters
was conducted.
Repeat shearing of DNA might be an option to avoid
wastage of DNA. Samples from rare species, clinical
samples and histological fixed sample are precious as the
quantity of nucleic acid extracted is low. As the results of
repeat shearing have not been previously reported, three
previously sheared DNA samples (no. 4, 6 and 7) were
selected for repeat shearing for an additional 1 minute.
Samples no. 4 and 7 (Figure 3a) showed a shift of the peak
and size distribution, from 206 bp to 180 bp and from 205
bp to 199 bp, respectively. Although improvement was
observed the peak size was still skewed to the right, slightly
out of the target range of 150 bp to 200 bp. The fragment
size distribution remained broad. Sample no. 6 on the other
hand, resulted in a peak size shift from 240 bp to 178 bp.
The distribution curve of fragment sizes for this sample was
sharp and fitted nicely into the target range of 150 bp to
200 bp (Figure 3b). Although good results were observed
Optimization of DNA shearing by sonication
203
Table 2. Peak sizes of ten genomic DNA samples extracted
from mononuclear cells of human blood, determined from
Bioanalyzer electropherograms.
Sample number
Peak size (bp)
1
2
3
4
5
6
7
8
9
10
Average
186
194
199
206
223
240
205
199
199
202
205.3±15.4 bp
in sample no. 6, repeat shearing of sheared DNA is still
not recommended as it seems to create a lot of off-target
fragments that will broaden the fragment size distribution
(shown in Figure 3a, graph region from 200 bp to 700 bp).
Increasing treatment time on the other hand may help
resolve this problem. As the DNA shearing process is
rate-limiting, fragment size generation, defined by
mean peak base pair size, can be affected by treatment
duration. A longer duration will produce smaller fragments
(Dibya et al., 2012; Schoppee and Wamhoff, 2011). Thus,
treatment time was extended by 20 seconds from the
recommended time in processing five samples of intact DNA. The results however, did not show much
alteration from original average peak base pair size
Figure 2. Electropherogram of sheared DNA run on a DNA 1000 Bioanalyzer.
Fragmentation procedures were conducted as recommended by the manufacturer. The two
small peaks on the far left and right of the graph were the upper and lower markers of
DNA ladder. Six overlaid samples (indicated by different colored linear plots) show the
distributions were outside the 150 bp to 200 bp range. Fluorescent unit (FU) on the y-axis
is indicative of DNA concentration.
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Optimization of DNA shearing by sonication
Figure 3. Electropherograms after repeat shearing of DNA sample. a) Repeat
shearing improved fragment size from 206 bp and 205 bp of sample no.4 and no.7 to 180
bp and 199 bp. However, off-target fragments were still present (region from 200 bp to 700
bp). b) repeat shearing improved fragment size of sample no.6 from 240 bp to 178 bp. The
region in green box indicates the target peak size range 150 bp to 200 bp. Fluorescent unit
(FU) on the y-axis is indicative of DNA concentration.
(201.4±17.2 bp vs 205.3±15.4 bp). The overlaid
electropherograms of five sheared DNA samples are shown
in Figure 4. Longer treatment times may help to achieve
the target peak size, but this is not recommended as it may
cause a broader peak size distribution and accumulation of
small fragments by breaking down more DNA. Moreover, a
prolonged acoustic sonication treatment time may
increase the temperature and affect the stability of the DNA
sample, and a treatment time of more than 370 seconds
(recommended time is 350 seconds) is very time
consuming especially when preparing a pooled NGS
library that consists of a large number of samples.
Peak incident power represents the power (Watts) emitted
from the transducer during each cycle (Covaris, 2011). The
Covaris shearing treatment consists of very high frequency
acoustic signals emitted in a series of bursts with each burst
being followed by a zero power state. Cycles per burst in
the setting indicates the number of acoustic oscillations
contained in each burst. Figure 5 shows the electropherograms of two intact samples after increasing shearing power
to 70W and 75W. Increase in peak incident power enhanced
the shearing effect thus achieving fragments in the target
range of 150 bp to 200 bp from both samples.
Lastly, the ability to reproduce target peak sizes
using a peak incident power of 75W was confirmed
using ten new intact DNA samples with the achievement
of an average peak size of 175±4.6 bp. Figure 6 shows the
electropherogram of seven overlaid samples and all the peak
sizes were within the range of 150 bp to 200 bp. Off-target
fragments (those appearing to be larger than 200 bp on
the electropherogram) produced as a result of incomplete
shearing were comparatively less common than from the
previous setting. Peak incident power and treatment time
were described as key factors in shearing bacterial genomic
DNA by Jeannotte et al. (2014). A 175W peak incident
power and 180 seconds of treatment time were identified
to be optimal values for shearing bacterial genomic DNA by
Covaris E220, though this differs somewhat from the more
cost-effective M220 model that we used.
As the length of adaptor sequence is constant in NGS
library preparation, the size of the DNA fragment from the
library which is inserted between the adapter sequences,
determined by the shearing step, becomes very important.
Optimal insert size is determined by the limitation of the
NGS instrumentation and by the specific sequencing
application. Sequencing fragments that do not fall in
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Optimization of DNA shearing by sonication
205
Figure 4. Overlaid electropherograms of five DNA samples extracted from
mononuclear cells of human blood with an extended 20 second treatment time. The
extended time did not produce a significant difference in the peak size. Fluorescent unit
(FU) on the y-axis is indicative of DNA concentration.
the recommended size distribution may cause low read
depth or decrease in the coverage of specific regions of the
sequence (Head et al., 2014). For the Illumina NGS platform,
insert size impacted the process of cluster generation
(library denaturation, dilution and distribution on the
two-dimensional surface of the flow-cell followed by
amplification of the library) by means of amplification
efficacy i.e. shorter products amplify more efficiently than
longer products (Sakharkar et al., 2004). In the case of
SureSelect target enrichment or other target enrichment
systems, fragment size distribution can affect the final
enrichment efficiency and the percentage of on-target
capture by the probes which will affect the final results and
quality of sequencing.
Fragmentation to the size range to suit the
requirements of different NGS platforms is also crucial to
avoid sequencing bias, as when the fragmented DNA are too
variable in size, the smaller off-target fragments tend to
Figure 5. Electropherograms of two DNA samples, extracted from mononuclear
cells of human blood, produced using peak incident powers of 70W and 75W.
Sample 1 is represented in a) and b), and sample 2 in c) and d). a) 70W, peak size: 186 bp,
b) 75W, peak size: 180 bp, c) 70W, peak size: 185 bp, and d) 75W, peak size: 178 bp.
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Optimization of DNA shearing by sonication
Figure 6. Overlaid electropherograms of seven sheared DNA samples, extracted from
mononuclear cells of human blood, produced using peak incident power 75W. All
sheared DNA samples produced a peak size in the target range. The green box indicates the
target peak size range of 150 bp to 200 bp.
be amplified more easily, resulting in over-representation
of these fragments. Thus, the optimization of the range of
insert sizes is important to minimize amplification
bias (Aird et al., 2011). Apart from the instrumental
considerations, the structure and GC content of a DNA
sample may also affect the fragmentation process which
eventually will lead to an uneven coverage of reads due to
bias in GC-poor or GC-rich sequences (Kozarewa et al.,
2009; Poptsova et al., 2014). Breaking of double-stranded
DNA by sonication occurs preferentially in 5’-CpG-3’
dinucleotides (Grokhovsky, 2006).Differential cleavage
rates (CpG> CpA/T > CpC) mean that the efficacy of
sonication for DNA fragmentation is sequence dependent,
determined by the CG content (Grokhovsky et al., 2011;
Dohm et al., 2008). However, the precise effect of GC
content on the quality of NGS data needs to be further
examined.
can be produced, results which were reproducible across
multiple samples. This optimized method can therefore be
useful when small fragments are required in NGS library
preparation.
CONCLUSION
Aird, D., Ross, M.G., Chen, W., Danielsson, M., Fennell,
T., Russ, C., Jaffe, D., Nusbaum, C. and Gnirke, A.
2011. Analyzing and minimizing PCR amplification
bias in Illumina sequencing libraries. Genome Biology
12:R18.
To initiate the preparation of an NGS library, the
production of good quality DNA (with regards to A260/280,
A260/230 ratios and integrity) is crucial, and several
parameters and techniques should be used to obtain the best
results. Fluorometry is a more accurate method to quantify
DNA than spectrophotometry. A higher input of DNA is
recommended in order to counter the loss of DNA that
occurs during shearing. Repeating or increasing the length
of DNA shearing treatment is not recommended as there
are disadvantages to these methods. We conclude that peak
incident power is the key factor in achieving a small target
peak without accumulating too many smaller fragments.
By increasing the peak incident power to 75W, a greater
proportion of fragments in the target 150 bp to 200 bp range
ACKNOWLEDGEMENTS
This study was supported by the Malaysia Genome
Institute, Ministry of Science, Technology and Innovation.
We would like to acknowledge the contribution of Irni
Suhayu Sapian and Norfarazieda Hassan. We would also like
to thank all volunteers for their participation.
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