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Improving Enzymatic DNA Fragmentation for
Next Generation Sequencing Library Construction
by Lynne Apone, Ph.D., Eileen Dimalanta, Ph.D., and Fiona Stewart, Ph.D., New England Biolabs, Inc.
INTRODUCTION
The Human Genome Project (HGP), which was
officially completed in 2003, was considered
to be one of the world’s largest collaborative
projects of its time (1). This involved many
research groups worldwide and had the lofty
goal of deciphering all 3 billion bases of thehuman genome. The project cost almost $4
billion dollars and took 13 years to completewith the available technology. Over a decade
later, advancements in next generation sequencing(NGS) technologies have enabled sequencing ofa human genome to become routine, taking lessthan two days, and at a tiny fraction of the costof the original HGP.
The ability to quickly and inexpensively sequence
whole genomes has truly revolutionized genomics
research. Where once single genes or familiesof genes were studied, now whole genomes,
exomes, transcriptomes and epigenomes areinterrogated. With recent advances, such as theability to multiplex and sequence many samples atonce, NGS has transitioned from a basic researchtool into the clinic, where it impacts discovery,
diagnostics and treatment of disease.
Advances in genomics driven by NGS, aswell as advances in the technology itself,
continue at an amazing pace and move us
closer to the realization of personalized
medicine, where clinical decisions are tailored
to an individual’s genome. However, if thispace is to continue, advances in all aspectsof the technology must also continue. This
includes early steps of the sequencing workflow,
specifically in the preparation of samples, before
they are sequenced.
To date, there are no sequencing platforms thatcan sequence intact DNA. Therefore, prior tosequencing, DNA molecules must be fragmented,
or broken, into smaller pieces. These DNA
fragments are then converted into libraries, bydifferent methods depending on the sequencing
platform to be used (Figure 1). In all cases, thelibraries generated consist of the fragments of
the unknown DNA to be sequenced, flanked
by pieces of known DNA (adaptors), which are
specific to each sequencing platform.

DNA FRAGMENTATIONAPPROACHESOne of the major bottlenecks to sample prep isthe first step: DNA fragmentation.
The size of the DNA fragments generated
depend on the sequencing platform being used,and can range from several hundred base pairs

FIGURE 1:
 raditional library preparation workflow
T

Extract
DNA

Fragment
DNA

PrepareLibrary

for short read sequencing technologies (e.g.,
Illumina®, Ion Torrent™) to >10 kb pieces
for long read sequencing technologies (e.g.,
Pacific Biosciences® and Oxford Nanopore
Technologies®). Methods for fragmenting
DNA are broadly split into two basic categories:
mechanical and enzyme-based. Mechanicalshearing methods include acoustic shearing,hydrodynamic shearing and nebulization, while
enzyme-based methods include transposons,
restriction enzymes and nicking enzymes.
Although many different options exist to
fragment DNA, final fragment size, amount ofstarting material, upfront capital investment, andscalability must be considered when choosing a
fragmentation method. Critically, in order to be
useful for NGS, the method used must shear the
DNA sufficiently randomly, so that the library
being sequenced is fully representative of theoriginal sample.

Mechanical Shearing
Options for mechanical fragmentation of DNA
range from small plastic nebulizer devices tosophisticated electronic instruments. The mostcommonly used technique utilizes focused

Amplify(optional)

Sequence

AnalyzeData

acoustic shearing devices, such as the instruments
made by Covaris®. This involves focusedtransmission of high-frequency, short wavelength
acoustic energy on the DNA sample. The size of
the DNA fragments generated (150 – 5,000 bp) is
controlled by changing both the intensity and theduration of the acoustic waves, and the protocols
used are the same regardless of the amount or GC
content of the DNA. Cost, challenges of scalability
and sample loss (often caused by sample transfer
after shearing), are some of the reasons that usersof this method seek alternatives, especially as
throughput increases.
If larger DNA fragments are required,hydrodynamic shearing can be used. In thismethod, hydrodynamic shear forces are appliedby pushing DNA through the small orifice ofa syringe. Size is controlled by altering the
speed at which the DNA is pushed through thesyringe. Centrifugation can also be used to createhydrodynamic force, by pulling the DNA sample
through a hole with a defined size. Here, the rateof centrifugation determines the degree of DNA
fragmentation. DNA fragments generated withhydrodynamic shear forces are typically in the

DNA fragmentation landscape prior to NEBNext Ultra II FS
Focused acoustic shearing

Enzymatic fragmentation•

Historical “gold standard”•

Scalable•

Simple control of fragment size•

Flexible reaction volumes•

Same protocol regardless of
GC content or input amount•

Ease of use•

Minimized DNA damage•

Prone to sequence bias•

May be sensitive tocontaminants in the
DNA sample•

Significant
optimization may
be required•

Transposases canhave very specificinput requirements•

Significant upfront instrument cost•

Significant disposables
cost and waste generation(tubes/plates)•

Time-consuming•

Challenging to scale•

Inflexible reaction volumes•

Prone to DNA damage

1

range of 1-75 kb, but require large DNA input
amounts (> 1 µg) and throughput is low.

Enzymatic Fragmentation
Enzyme-based fragmentation of DNA is an
attractive alternative to mechanical shearing
methods, as it does not require upfront capital
expense, is amenable to quickly processing many
samples at the same time, and reduces sample
loss. Historically, the main concern with thismethod has been sequence bias, as many enzymes
that cleave DNA have recognition sequences orsequence preferences.
Transposases fragment DNA by cleaving andinserting a short double-stranded oligonucleotide
to the ends of the newly cleaved molecule. The
inserted oligonucleotide must contain a sequencethat is specific to the particular transposase being
used. While this method is fast and has low input
requirements, the known sequence bias associatedwith transposases make them incompatible withsome applications.
The great majority of restriction enzymes havevery specific recognition and cleavage sites, andtherefore are not suitable for the random cleavage
required for most NGS applications. However,restriction enzymes do have utility in a workflow
called RAD-Seq (Restriction-site Associated DNA
Sequencing). Here, the sequence bias from therestriction enzyme cut site is exploited to target
certain regions for sequencing.
Lastly, non-specific nicking enzymes can beused to fragment DNA. These enzymes have lesssequence bias than transposases or restriction
enzyme-based methods, and generate fragmentsof different sizes (generally 50 bp-1 kb) in atime-dependent manner: the longer the reaction
time, the smaller the fragments. Historically,
these enzymes have required significant reaction
optimization. They have been sensitive tothe buffering conditions of the DNA sample,
and required different reaction conditions fordifferent DNA input amounts and for varying
GC content. While appealing in theory, thishas made enzymatic fragmentation methods
more challenging to implement, especially inlaboratories where a variety of sample types andamounts are used.

Emerging drawbacks to the gold standard
Of all the techniques described, acoustic shearing
has traditionally been the method of choice
for short read sequencing technologies, such as
Illumina; its popularity a result of robust shearing
with low bias. However, this method requires a

Fragmentation/
End Repair/dA-Tailing

AdaptorLigation

Clean up/Size Selection Amplification

Clean upsignificant financial investment in equipment,
which can be prohibitive for many researchers.
In addition, throughput is low and sample loss
high, creating bottlenecks and limitations forusers of the technology. Interestingly, recent
studies have also shown that oxidative damage
can occur during mechanical shearing processes(2,3).

Importantly, enzymatic shearing of DNA with thismethod does not introduce bias into the library,
and this method is suitable for input DNA witha full range of GC content. Reduced sample loss
and increased efficiencies of the workflow enable
use of lower input amounts, with a range of 100
pg – 0.5 μg, and insert sizes of 100 bp to 1 kb
can easily be generated.

IMPROVING DNA
FRAGMENTATION FOR NGSLIBRARY CONSTRUCTIONPERFORMANCE
Increased Library Yields

To address the challenges associated withexisting fragmentation approaches, NEB
has developed a fragmentation system, theNEBNext® Ultra™ II FS DNA Library Prep
Kit (NEB #E7805, E6177), in which a unique
enzymatic fragmentation reagent is fully
integrated into library preparation to generate
low bias, high quality NGS libraries, with asimple, scalable workflow (for more information
see page 6).
In order to reduce the NGS sample prep
bottleneck, improvements in both performanceand ease of use were necessary. In this work,
we have focused on the DNA fragmentation
step. Our new DNA fragmentation reagent
is combined with end repair and dA-tailing
reagents, and subsequent adaptor ligation is also
carried out in the same vial (Figure 2). For low
input samples, PCR amplification is performed
prior to sequencing.

The use of enzymatic fragmentation can result inhigher library yields as compared to mechanicalshearing workflows, as the latter results in sample
loss and DNA damage. Achieving sufficient libraryyields for high quality sequencing from very
low input amounts can be especially challenging
with mechanical shearing of DNA, a situation
compounded by the preference to amplify
libraries using as few PCR cycles as possible.
Integration of our unique fragmentation reagent
with end repair and dA-tailing, removing sample
cleanup prior to ligation and eliminating multiple
transfer steps all help to minimize sample loss.
When combined with the high reaction efficiencesof each step in the workflow and lack of DNA
damage cuased by mechanical shearing, NEBNext
Ultra II FS generates higher yields than library
preparation using mechanical shearing methods.
High library yields can be achieved with input

FIGURE 3:
 EBNext Ultra II FS DNA produces higher yields ofN

PCR-free libraries
Libraries were prepared from Human NA19240 genomic DNA using the input amounts shown, without amplification.
NEBNext Ultra II FS libraries were prepared using a 20-minute fragmentation time. “Covaris” libraries were preparedby shearing each input amount in 1X TE Buffer to ~200 bp using a Covaris instrument, followed by library construction
using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Library yields were determined by qPCR using the
NEBNext Library Quant Kit for Illumina (NEB #E7630). Error bars indicate standard deviation for an average of 2 libraries.
60

NEBNext Ultra™ II FS®

Covaris®

50

Library Yield (nM)

Nebulization is another method used tomechanically fragment DNA. Nebulization uses
compressed air to force DNA through a small
hole in a nebulizer unit and DNA fragment size
is determined by the pressure used. Although
this method is inexpensive and fragment size issomewhat tunable (typically 700 – 5000 bp insize), microgram quantities of DNA are required
for starting material, and the method is mostsuitable for small numbers of samples.

FIGURE 2:
 EBNext Ultra II FS Kit workflow
N

40
30
20
10
0

50 ng

100 ng200 ngDNA Input

500 ng2

amounts as low as 100 pg of human genomic
DNA with amplification, or as low as 50 ng for
PCR-free workflows (Figure 3, page 3).

Improvements in Library Quality
As mentioned above, a historical concern
regarding the use of enzymatic fragmentation
methods was the potential for introduction ofbias into a sequencing library, and ultimately
into the sequencing data. As shown, this new
fragmentation reagent provides consistentuniformity of GC coverage at the full range of
input amounts (Figure 4) and over a broad rangeof GC composition (Figure 5).

EASE OF USE

In addition, oxidative damage markers typicallyassociated with mechanical-shearing methods(2,3) are observed at significantly lower frequency
in libraries made using this new fragmentation
system (Figure 6, page 5). Importantly, a greater
difference between the observance of thesemarkers is seen with lower input amounts.
This highlights the higher quality of libraries
constructed with this new method compared to
Covaris-sheared DNA libraries, especially at low
input amounts.

Robustness of DNA Fragmentation
Consistent and reliable fragmentation is criticalfor a new method to be adopted. We optimized
this new fragmentation system to be insensitive
to variables such as input amounts, GC content,
and DNA buffer conditions. In practice, thesedetails are often unknown for a sample, requiring
clean up and quantification prior to traditional
enzymatic DNA fragmentation methods.
Even when all of the variables are known,
traditional enzymatic methods require different
fragmentation parameters for each type of sample
and DNA input amount. This new fragmentation
system addresses all of these issues by requiring
just a single-fragmentation protocol for thefull range of input amounts (100 pg – 0.5 μg)(Figure 7, page 5) and for the full range of GC
content (Figure 5). Also, input DNA can be inwater, Tris, 0.1X TE or 1X TE (Figure 8, page5). Fragmentation using the new system is timedependent, and final library sizes ranging from100 bp – 1 kb can be generated by simply
changing the fragmentation time.

FIGURE 4:
 EBNext Ultra II FS DNA provides uniform GC coverage withN

human DNA over a broad range of input amounts
Libraries were prepared from Human NA19240 genomic DNA using the input amounts and number of PCR cycles shown, and a
20-minute fragmentation time was used. Libraries were sequenced (2 x 76 bp) on an Illumina® MiSeq®. Reads were mapped to thehg19 reference genome using Bowtie 2.2.4 and GC coverage information was calculated using Picard’s CollectGCBiasMetrics (v1.117).
Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each GC% is indicated by
the vertical grey bars, and the colored lines represent the normalized coverage for each library.
2.0

1.6

Coverage (Normalized)

CONCLUSION

500 ng (3 cycles)
100 ng (4 cycles)
50 ng (5 cycles)
1 ng (9 cycles)
500 pg (10 cycles)
100 pg (13 cycles)

1.8

1.4
1.2

The continued expansion of the use of next
generation sequencing depends in significant parton overcoming the limitations and bottlenecks
associated with high-quality library preparation,
including the initial DNA fragmentation step.
While acoustic shearing has for some time been
the method of choice for NGS, limitations interms of instrumentation, throughput and sample
damage necessitate sourcing an alternative

1.0
0.8
0.6
0.4
0.2
0

0

5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

GC Percentile

FIGURE 5:
 EBNext Ultra II FS DNA provides uniform GC coverage for microbial DNA over a broad range of GC
N

composition
Libraries were prepared using 1 ng of a mix of genomic DNA samples from Haemophilus influenzae, Escherichia coli (K-12 MG1655) and Rhodopseudomonas palustris, with 9 PCR cycles, and sequenced on an
Illumina MiSeq. NEBNext Ultra II FS libraries were prepared using a 20-minute fragmentation time. “Covaris” libraries were prepared by shearing 1 ng of DNA in 1X TE Buffer to an insert size of ~200 bp usinga Covaris instrument, followed by library construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Reads were mapped using Bowtie 2.2.4 and GC coverage information was calculated using
Picard’s CollectGCBiasMetrics (v1.117). Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each GC% is indicated by the vertical grey bars, and thecolored lines represent the normalized coverage for each library.

NEBNext Ultra™ II FS

Covaris

®

2.0

2.0

1.5

1.5
1.0

H. influenzae(38% GC)

0.5
2.0
1.5
1.0

E. coli(51% GC)

0.5
2.0
1.5
1.0

H. influenzae(38% GC)

0.5
2.0
1.5
1.0

E. coli(51% GC)

0.5
2.0
1.5
1.0

R. palustris(65% GC)

0.5
0Coverage (Normalized)

Coverage (Normalized)

1.0

®

0

10

20

30

40

50

60

70

80

90

100

R. palustris(65% GC)

0.5
0

0

10

20

GC Percentile

Kapa™ HyperPlus

30

40

50

60

GC Percentile

Illumina Nextera XT
®

®

70

80

90

100

3

FIGURE 6:
 EBNext Ultra II FS DNA libraries show reduced markers of oxidaN

solution for many users. This new method for
enzymatic DNA fragmentation and library
preparation addresses these issues, further
streamlines the process and improves the qualityof NGS libraries. The broadening of the input
amount range to as low as 100 picograms enables
access to high-quality sequencing of samples
not achievable by previous methods, and thereliability and ease of use of the method not only
allows automation, but also successful adoption
by users with a wide range of laboratory skills.

tive damage compared to libraries produced by mechanical shearing
Libraries were prepared from 1 ng and 100 ng Human NA19240 genomic DNA, using 9 and 4 PCR cycles, respectively. NEBNext Ultra II FS
libraries were prepared using a 20-minute fragmentation time. “Covaris” libraries were prepared by shearing each input amount in 1X TE Buffer
to ~200 bp using a Covaris instrument, followed by library construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries
were sequenced on an Illumina® HiSeq® 2500 (2 x 75 bp). 723M reads were randomly sampled (seqtk) and aligned to the GRCh38 fullreference genome using bwa (0.7.15). Adaptors were trimmed prior to alignment using trimadap (r9). Duplicates were marked using samblaster(0.1.24). Variants were called on chromosome 1 using freebayes (1.0.2.29) with frequency based options requiring at least 10 reads per site.
More variants are observed for C>A and G>T transversions compared with all other variants for PCR-amplified Covaris libraries. These variants
indicative of oxidative damage are not pronounced in NEBNext Ultra II FS libraries.
Other Variation
500

Oxidative
DamageSignature
Variation®

CovarisOther Variation

Oxidative
DamageSignature
Variation

References:
1. https://www.battelle.org/docs/default-source/misc/battelle2011-misc-economic-impact-human-genome-project.pdf
2. M. Costello et al. (2013) Nucleic Acids Research, 41, e67.
3. L. Chen, et al. (2017) Science, 17, 355: 752-756.

400

Number of Variants Detected

300
200
100
0
400

®

NEBNext Ultra™ II FS

300
200

1 ng

G>T

C>A

T>C

T>G

T>A

G>A

G>C

C>T

C>G

A>T

A>G

A>C

G>T

C>A

T>C

T>G

T>A

G>C

C>T

G>A

C>G

A>T

A>G

0

A>C

100

100 ng

FIGURE 7:

FIGURE 8:

NEBNext Ultra II FS DNA provides consistent
fragmentation regardless of input amount

NEBNext Ultra II FS DNA provides consistent
fragmentation of DNA in water, Tris or TE

Libraries were prepared from Human NA19240 genomic DNA using the input amounts shown. NEBNext
Ultra II FS libraries were prepared using a 20-minute fragmentation time. Library size was assessedusing the Agilent® Bioanalyzer®. Low input (1 ng and below) libraries were loaded on the Bioanalyzer
without a dilution. High input libraries were loaded with a 1:5 dilution in 0.1X TE.
[FU]

Libraries were made using 100 ng Human NA19240 genomic DNA using the NEBNext Ultra II FS kit or
the Kapa HyperPlus Kit. Fragmentation conditions targeting ~200 bp inserts were used, which would
generate ~320 bp libraries. For the NEBNext Ultra II FS kit, input DNA was in H2O, Tris, 0.1X TE or 1X
TE . For the Kapa HyperPlus kit, libraries were made using the recommended dilution of the supplied
Conditioning Solution (CS), or using DNA in Tris, 0.1X TE or 1X TE, in the absence of either Conditioning Solution or 3X bead clean up. Library size distribution was assessed using the Agilent Bioanalyzer.
Fragmentation is consistent for the NEBNext Ultra II FS kit for DNA in H2O, Tris, 0.1X TE or 1X TE.

100 pg500 pg
1 ng
50 ng
100 ng200 ng300
250®

NEBNext Ultra™ II FS

200

Water

150

7000

100

2000

Tris

0.1X TE 1X TE

Kapa™ HyperPlus
CS

Tris

0.1X TE 1X TE

1000

Size (bp)

50
0

35

100

200

300

400 500 600 1,000 2,000

10,380

[bp]

600
500
400
300
200
150
100
35

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Inc. For more information, please email us at gbd@neb.com. The use of these products may require you to obtain additional third
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AGILENT®, BIOANALYZER® and CLEARSEQ® are registered trademarks of Agilent Technologies. ILLUMINA®, HISEQ®, MISEQ® and
NEXTERA® are registered trademarks of Illumina, Inc. COVARIS® is a registered trademark of Covaris, Inc. BIO-RAD® is a registeredtrademark of Bio-Rad Laboratories, Inc. KAPA™ is a trademark of Kapa Biosystems.
© Copyright 2017, New England Biolabs, Inc.; all rights reserved.

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