Adapters play a crucial role in next-generation sequencing (NGS). They enable DNA fragment binding to flow cell hardware and can be used to identify sequences in multiplexed library analysis. However, adapters can be prone to dimerization, which can result in inaccurate data, decreased quantification precision, suboptimal flow cell loading and compromised genome coverage.
This technical overview highlights the latest fragment analysis and electrophoresis solutions for adapter dimer detection. It explores how greater detection and resolution capabilities can support sequencing efficiency and quality control (QC).
Download this technical note to discover:
- The crucial role of adapters dimers in NGS library preparation workflows
- A preoptimized method for adapter dimer detection
- Ways to improve QC and decision making in your NGS applications
An NGS library is the compilation of many DNA fragments pooled together for
subsequent analysis. During the process of library preparation, DNA fragments
are ligated to a known sequence, or adapter. These adapters allow the DNA
fragments to bind to the flow cell during the sequencing run. The adapter is
designed to include a primer binding site to initiate sequencing. Additionally,
adapters can include unique sequences that can be used as indexes to help
distinguish between multiplexed libraries during data analysis. However,
sometimes two adapters may ligate to each other without an insert, forming
a dimer that can be amplified, bind, and cluster on the flow cell, generating
erroneous sequencing data (Figure 1). Even small amounts of adapter dimers
in a library can contribute to detrimental sequencing results. The presence
of adapter dimers can affect the accuracy of library quantification, leading
to suboptimal flow cell loading and reducing clustering efficiency. Smaller
fragments such as dimers are also preferentially sequenced over larger
fragments, so even low levels of adapter dimers could contribute to issues such
as reduced output, lower diversity, and decreased genome coverage1,2. Just 5%
of adapter dimer contamination in a library can cause up to 50% of the reads to
be adapter sequences3
. Thus, it is highly recommended to minimize and remove
adapter dimers from the library before sequencing.
Detection of Adapter Dimers in NGS
Libraries with the Agilent Fragment
Analyzer and TapeStation Systems
While the optimal amount of adapter dimer in a library is
zero, there are thresholds of adapter dimer in a library that
may be present and still generate satisfactory sequencing
results. As an example, random flow cell technologies from
Illumina, including the MiniSeq and MiSeq, recommend that
no more than 5% of the library be composed of adapter
dimers. Patterned flow cells, such as the Illumina HiSeq X
and NovaSeq 6000, are more sensitive and recommend a
threshold of only 0.5% dimer4
. Quality control (QC) steps are
crucial to determining if a library contains any adapter dimers.
The Agilent automated electrophoresis portfolio offers a
suite of instruments that allow for accurate and reliable QC
throughout NGS library preparation.
This technical overview highlights the sensitivity and
resolution of the Agilent Fragment Analyzer and TapeStation
systems to detect adapter dimers even lower than the
recommended threshold of 0.5% and deliver an accurate
assessment of the percentage of adapter dimer in the library.
This information provides valuable data to evaluate the quality
of the library and enables researchers to make important
decisions regarding subsequent analysis.
NGS Library Preparation Schematic
Figure 1. During the process of NGS library preparation, known DNA adapter
sequences are ligated to the 5’ and 3’ ends of the DNA. Generally, one
adapter will contain the primer sequence, while the other is used to bind the
library to the flow cell for sequencing. Adapter dimers form when the two
adapters ligate to each other instead of the target insert.
A DNA library was prepared using the NEXTflex™ Rapid DNASeq Kit (Bioo Scientific, part number 5144-01). 0.8x SPRI bead
cleanup was performed two times to eliminate any inherent
dimers. An RNA library was prepared using the NEBNext Ultra
RNA Library Prep Kit for Illumina (NEB, part number #E7530),
following manufacturer’s protocol.
A 150 bp NoLimits Fragment (Thermo Fisher Scientific)
at 50 pg/µL was utilized to mimic an adapter dimer peak
(hereafter referred to as adapter dimer). The fragment was
added to aliquots of the NGS libraries such that the library
concentration stayed consistently at 500 pg/µL and contained
varying amounts of the 150 bp fragment: 0, 0.1, 0.2, 0.5, 1, and
5%. Controls were prepared for each fragment amount using
1x TE instead of library.
Samples were analyzed using Agilent automated
electrophoresis instruments. Analysis on the Agilent 5200
Fragment Analyzer system was performed with the Agilent
HS NGS Fragment kit (1-6000 bp) (part number DNF-474) and
the Agilent HS Small Fragment kit (part number DNF-477).
Samples were analyzed on the Agilent 4200 TapeStation
system using the Agilent High Sensitivity D1000 ScreenTape
(part number 5067-5584 and 5067-5585) and Agilent High
Sensitivity D5000 ScreenTape (part number 5067-5592 and
The Agilent ProSize data analysis software was utilized to
analyze the samples run on the Fragment Analyzer. The
baseline was adjusted as necessary to ensure full integration
of the smear sample. The minimum peak height was adjusted
to 10 RFU, and the peak width to 40 seconds. Likewise, the
TapeStation analysis software was used to analyze the
samples run on the TapeStation. The peak width of the library
was manually adjusted by moving the start and end points
to include the entire smear and enable accurate percent
integrated area calculations. Fragments that were visualized
but not automatically detected were manually added as a
peak. The sensitivity of both systems was analyzed based on
their ability to visualize the fragment and to accurately report
the percent concentration of the fragment peak compared to
the total sample.
DNA fragments Adapters
NGS Library Adapter dimers
Results and discussion
Detection sensitivity of adapter dimer
The NGS library preparation workflow includes several
instances at which QC checks are recommended, including
evaluation of the integrity of both the starting sample and the
final library prior to sequencing. One reason for this final QC
step is to ensure that the library is devoid of small fragments
such as adapter dimers, as they can significantly affect the
sequencing data. Different sequencing platforms have a
range of dimer that can be tolerated without interfering with
the sequencing results. An acceptable threshold of dimer
contamination can range from 5% to as little as 0.5%. The
Fragment Analyzer and TapeStation systems can be used
to detect and quantify very small amounts of dimer present
within different types of NGS libraries. Each instrument uses
various analysis kits and assays to assess samples covering
a broad range of sizes and concentrations (Table 1).
Typically, short read NGS libraries are within a size range
that is appropriate for analysis using the HS NGS kit for the
Fragment Analyzer systems or the HS D5000 ScreenTape
assay for the TapeStation systems. The HS NGS kit offers
accurate sizing from 100 to 6,000 bp, while the HS D5000
ScreenTape assay ranges from 100 to 5,000 bp. In this
example, an NGS library with a target insert size of 150 to
250 bp was analyzed with both systems and shown to have
a peak size of about 370 bp following adapter ligation, with
a distribution ranging from approximately 250 to 2,000 bp.
After cleanup with SPRI beads, the library was absent of
any inherent primer or adapter dimer. To assess the ability
of the Fragment Analyzer and TapeStation to detect small
percentages of adapter dimer, a 150 bp fragment was
added to the library at 0.1% up to 5% of the concentration of
the library. Each sample was analyzed on both systems in
The ProSize data analysis software utilized with the Fragment
Analyzer displays the results in both a digital gel format and
an individual electropherogram of each sample. A Peak Table
also records information about the sample, including the size,
concentration, and percentage of each peak. Each sample
is automatically analyzed, but the user can adjust settings
such as the peak width, minimum peak height, and baseline
setpoints to ensure that each peak is appropriately integrated.
Figure 2 shows representative electropherogram images of
the NGS library containing varying percentages of a 150 bp
fragment from the Fragment Analyzer using the HS NGS kit.
The adapter dimer fragment was detected at all variations
tested, from 5% (Figure 2A) to 0.1% (Figure 2D). The percent
total reported by the Fragment Analyzer was accurate for the
1%, 0.5%, and 0.1% samples, at 0.9%, 0.5%, and 0.1%. The
adapter dimer fragment at 5% was reported at 4.2% of the
total library concentration (Table 2). Additionally, the Fragment
Analyzer displayed accurate sizing of the fragment, with a
size of 153 or 154 bp in all sample wells and a percent error of
less than 3%, within the kit specification of 5%.
The TapeStation analysis software also displays a digital gel
image and electropherogram of each sample. The Peak Table
provides the size, concentration, and percent integrated area
for each peak. The width of a peak can be adjusted by moving
the start and end points of the peak to ensure integration of
the entire sample, and thus determine the accurate percent
integrated area for each peak. With the HS D5000 assay, the
TapeStation was able to detect the fragment at 5% to 0.5%
of the total concentration, as shown in Figure 3. The adapter
dimer fragment displayed an average size of 133 bp, slightly
smaller than the expected 150 bp, with a percent error of less
than 12%, within the kit specification of 15%. The percent
integrated area reported by the TapeStation was within
0.3 percentage points from the expected 5%, 1%, and 0.5%
samples (Table 2).
Instrument and Kit Sizing Range Sizing Accuracy Quantitative Range Quantitative Accuracy Resolution
HS Small Fragment kit
50 – 1,500 bp ±5% Smears: 100 – 5,000 pg/µL
Fragments: 5 – 500 pg/µL
±25% 50 – 900 bp: 5%
900 – 1,500 bp: 10%
HS NGS kit
100 – 6,000 bp ±5% Smears: 50 – 5,000 pg/µL
Fragments: 5 – 500 pg/µL
±25% 100 – 1,000 bp: 5%
1,000 – 6,000 bp: 10%
HS D1000 ScreenTape assay
35 – 1,000 bp ±10% 10 – 1,000 pg/µL
(sensitivity to 5 pg/µL)
±20% 35 – 300 bp: 15%
300 – 1,000 bp: 10%
HS D5000 ScreenTape assay
100 – 5,000 bp ±15% 10 – 1,000 pg/µL
(sensitivity to 5 pg/µL)
±25% 400 – 5,000 bp: 15%
Table 1.Specifications of the kits used with the Agilent Fragment Analyzer systems and Agilent TapeStation systems for NGS library quality control.
A. 5% Fragment B. 1% Fragment
C. 0.5% Fragment
Figure 2.Representative electropherograms from the Agilent 5200 Fragment Analyzer system of an NGS library containing known amounts of adapter dimers
using the Agilent HS NGS fragment kit. The 150 bp adapter dimer fragment can be visualized at A) 5%, B) 1%, C) 0.5%, and D) 0.1% of the total library concentration.
D. 0.1% Fragment
Fragment Analyzer HS NGS Kit TapeStation HS D5000 ScreenTape Assay
Expected Adapter Dimer Fragment Size (bp) % (Conc.) Fragment Size (bp) % Integrated Area
5% 153 4.2% 124 4.7%
1% 154 0.9% 138 1.3%
0.5% 153 0.5% 138 0.8%
0.1% 154 0.1% Not detected Not detected
Table 2. Reported size and percentage of adapter dimer present within an NGS library analyzed on the Agilent 5200 Fragment Analyzer system and the Agilent
4200 TapeStation system.
A. 5% Fragment
B. 1% Fragment
C. 0.5% Fragment
Figure 3.Representative electropherograms from the Agilent 4200
TapeStation system of an NGS library containing known amounts of adapter
dimers using the Agilent HS D5000 ScreenTape assay. The adapter dimer
fragment can be visualized at A) 5%, B) 1%, and C) 0.5% of the total library
Resolution of adapter dimers
Depending on the type of sample being analyzed, the
automated electrophoresis systems offer a variety of kits
and assays for different sizing ranges and separation
resolution. Each assay utilizes different separation methods,
gel chemistries, and sizing ladders. To demonstrate the
differences in some of the kits that can be used for smaller
molecular weight samples, RNA-Seq libraries containing
varying amounts of adapter dimer were compared across
both systems, with two kits of similar sizing ranges for each
(Table 1). For the Fragment Analyzer, the samples were
analyzed on the HS Small Fragment kit and the HS NGS kit.
Samples were analyzed on the TapeStation using the HS
D1000 assay and the HS D5000 assay.
The RNA-Seq library preparation protocol used was optimized
to generate approximately 200 bp inserts. As shown in
the electropherogram images in Figure 4, the final library
distribution ranged from approximately 200 to 600 bp,
with the largest area of the curve at about 270 to 300 bp,
depending on the analysis kit. The library contains a small
amount of 130 bp dimer contamination, the 150 bp fragment,
and the library, which can be seen at approximately 130 bp.
This contamination was intentionally left in the sample, and
a 150 bp fragment mimicking adapter dimer was spiked into
the sample at varying amounts from 0.1% to 5% to examine
the resolution capabilities of the analysis kits.
Shown in Figure 4 are representative examples of the library
with a spike-in of 1% of the 150 bp fragment. The Fragment
Analyzer provided high-resolution separation of the inherent
130 bp dimer contamination, the 150 bp fragment, and the
library with both the HS Small Fragment (Figure 4A) and the
HS NGS kits (Figure 4B). Both kits also showed accurate
sizing and percent total calculations for the fragment from
0.2% to 5%, with the HS NGS kit also able to detect the
fragment at as low as 0.1% of the total concentration
The TapeStation HS D1000 assay fully resolved the 130 bp
dimer contamination, the 150 bp fragment, and the library
(Figure 4C) and was able to detect the adapter fragment
among the entire range from 0.1% to 5% spike-in (Table 3).
Alternately, the HS D5000 assay was only able to visualize the
fragment from the library at 5% of the concentration (Figure
4D). The concentration of the fragment in the 0.1% and 0.5%
samples was below the assay’s limit of detection of 5 pg/uL
and could not be resolved from the library smear.
A. Fragment Analyzer HS Small Fragment kit. 1% adapter dimer. B. Fragment Analyzer HS NGS kit. 1% adapter dimer.
C. TapeStation HS D1000 ScreenTape assay. 1% adapter dimer. D. TapeStation HS D5000 ScreenTape assay. 5% adapter dimer.
Figure 4.Representative electropherograms of an RNA-Seq NGS library with known amounts of adapter dimer contamination. The library containing 1% adapter
dimer was visualized on the Agilent 5200 Fragment Analyzer with A) the HS Small Fragment kit and B) the HS NGS kit, and on the Agilent 4200 TapeStation system
with the HS D1000 ScreenTape assay. D) 5% adapter dimer on the Agilent 4200 TapeStation system with the HS D5000 ScreenTape assay. The red arrow in each
figure points to the 150 bp NoLimits DNA fragment spiked into the library to mimic the adapter dimer.
Evaluation of the same samples across multiple assays and
instruments demonstrates the importance of choosing the
most appropriate analysis method for different applications.
The Fragment Analyzer and TapeStation data presented
here can help users decide which method of QC is best for
their samples depending on the type of sequencing they will
be performing and the amount of dimer that is considered
acceptable for their specific workflows.
Table 3. Reported size and percentage of adapter dimer present within an RNA-Seq NGS library analyzed on the Agilent 5200 Fragment Analyzer system and the
Agilent 4200 TapeStation system.
HS Small Fragment Kit
HS NGS Kit
HS D1000 ScreenTape Assay
HS D5000 ScreenTape Assay
% (Conc.) Fragment Size
% (Conc.) Fragment Size
% Integrated Area Fragment Size (bp) % Integrated Area
5% 151 4.6% 154 4.9% 161 4.3% 132 5.8%
1% 150 0.9% 154 1.0% 157 0.7% Not detected Not detected
0.5% 151 0.4% 154 0.5% 159 0.4% Not detected Not detected
0.2% 151 0.2% 154 0.2% 170 0.1% Not detected Not detected
0.1% Not detected Not detected 154 0.1% 166 0.1% Not detected Not detected
As sequencing platforms are becoming more sensitive,
steps must be taken to ensure that the sequencing libraries
contain a minimum of undesired products that could
impede the sequencing results. For example, the formation
of adapter dimers can reduce the amount of adapters that
are available to ligate to the target insert, resulting in fewer
usable sequencing reads. Additionally, the small adapter
dimer fragments present in the library will be preferentially
sequenced over the target. As such, QC of the final library
is vital to ensuring that the sample is of high integrity, the
correct size, and contains minimal amounts of primer or
This technical overview summarized the sensitivity and
resolution that can be achieved with the Agilent Fragment
Analyzer and the Agilent TapeStation by utilizing different
analysis kits. The Agilent Fragment Analyzer HS NGS kits
and the Agilent TapeStation HS D1000 kits both provide high
sensitivity necessary to detect adapter dimer in traditional
NGS libraries even lower than the 0.5% to 5% threshold
recommended by sequencing cores. The Fragment Analyzer
HS Small Fragment and HS NGS kits and the TapeStation
HS D1000 kit provides high resolution to enable separation
of low amounts of adapter dimer from the library smear. By
providing valuable data regarding the quality of NGS libraries,
the Agilent automated electrophoresis systems can help
researchers make important decisions about their sequencing
1. Adapter Dimers Causes, Effects, and How to Remove
Them. Illumina Knowledge article, publication number
2. How Short Inserts Affect Sequencing Performance.
Illumina Knowledge article, publication number 3874.
3. Frequently Asked Questions. Stanford Medicine, Genome
Sequencing Center. https://med.stanford.edu/gssc/faq.
html (accessed 2023-03-07).
4. Primer Dimer Sequencing Requirements. University of
North Carolina at Chapel Hill, Integrated Genomics Core.
FINAL.pdf (accessed 2023-03-07).
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2023
Published in the USA, June 1 2023