Application compendium - 2nd edition

Sample Quality Control in Next-Generation Sequencing Workflows
Agilent Automated Electrophoresis Portfolio
Introduction

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Introduction
Throughout the updated edition of this applicationcompendium, you will find various application notes
and technical overviews providing you essential
information for success in your nucleic acidsample quality assessment during next-generation
sequencing (NGS) workflows.

Sequencing

This collection of resources includes topics covering:– Why sample quality control is important in NGS workflows– How quality metrics help provide reliable assessment ofsample integrity– How to analyze and assess quality of cell-free DNA (cfDNA),
formalin-fixed paraffin-embedded (FFPE) DNA and RNA,
genomic DNA (gDNA), total RNA, and NGS library samples– Which instrument is most suitable for different workflows
and applications

Samplepreparation

Library
preparation

Targetenrichment

Input
sample QC

Intermediate
sample QC

Finallibrary QC

Recommended steps for quality control during NGS library preparation.

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Automated Electrophoresis Instruments to Support Your NGS Workflows
Next-generation sequencing is an essential tool in molecular biology laboratories for the analysis of nucleic acid samples
in numerous basic, translational, and clinical research settings. Quality control (QC) of input samples, at intermediate
steps of the preparation process, and of the final libraries before sequencing, can help save time and resources byidentifying samples that are of poor quality or of insufficient concentration to yield successful sequencing data.
The Agilent automated electrophoresis portfolio provides several instruments for QC of various nucleic acid sample
types to support your NGS workflow.
Discover which Agilent automated electrophoresis solution fits your application. Select an instrument below for more information.

TapeStation systems

Automated electrophoresis QC for various
workflows for the analysis of size, quantity, andintegrity of RNA and DNA input samples.

Fragment Analyzer systems

Femto Pulse system

Bioanalyzer system

Automated parallel capillary electrophoresis
providing accurate and reliable quality assessment
for different sample types with a broad range of kits.

Automated pulsed-field capillary electrophoresis
enabling exceptional QC for low concentration nucleic
acid samples and high-molecular weight DNA.

See how the Agilent Bioanalyzer system compares toour other automated electrophoresis instruments.

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Automation for Library Preparation and Target Enrichment
Prepare high-quality, sequencing-ready libraries with more consistency and lab efficiency using automated solutions
for library preparation and target enrichment. Choose between two automation platforms from Agilent, both of which
support enzymatic fragmentation of DNA, reverse transcription for RNA, and bead cleanup. These automation platforms
offer a Reagent Rental Program for SureSelect reagents to offset capital equipment expense.

Bravo NGS system

Simplified NGS workflow automation without sacrificing
quality, in a small footprint.

Bravo NGS workstation

High-throughput NGS sample preparation robot forincreased efficiency and maximized walkaway time.

Magnis NGS prep system

Compact benchtop platform providing an automated,completely walkaway NGS library preparation and target
enrichment solution.

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS Products For Reliable Results
Besides sample QC instrumentation and automation options, Agilent offers an extensive porfolioof NGS products that help you generate reliable data.
Agilent SureSelect hybridization capture-based reagents include custom and catalog kits forlibrary preparation and target enrichment to meet your sequencing needs. The Agilent Alissa
Clinical Informatics platform enables innovative NGS and CGH data analysis to optimize
workflows across technologies and applications. The platform seamlessly and efficiently
integrates raw read alignment, variant annotation and classification, and reporting modules.
See which products are best suited for your NGS-based research.

Explore the portfolio

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Agilent TapeStation Systems
The Agilent TapeStation systems offer
scalable throughput and rapid results, making
them the ideal solution for quality control ofbiological samples in NGS.
While the higher throughput Agilent 4200 TapeStation
system can analyze DNA and RNA samples froma 96-well plate, the smaller footprint Agilent 4150
TapeStation instrument is the lower throughput
alternative for analyzing up to 16 samples per run.
Both systems offer walk away operation with fully
automated sample processing.
Key benefits

No matter which system you choose, full assay
compatibility is guaranteed as both instruments
share the same ScreenTape consumables.

BarcodeAutomated assay detection

ScreenTape details
At-a-glance expiry dateBuffer chamber
Automatic sample loading

Individual sample lanes
Zero carry-overElectrodes
Individual sample separation

Overview of Agilent ScreenTape device.

Learn more about the TapeStation systems

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Agilent Fragment Analyzer Systems
The Agilent Fragment Analyzer systems use
automated parallel capillary electrophoresis (CE)to provide reliable quality control (QC) of nucleic
acids for various applications, including nextgeneration sequencing.

Key benefits

The 5200, 5300, and 5400 Fragment Analyzer systems
are used for low to high throughputs, breaking throughanalytic bottlenecks and streamlining nucleic acidanalysis workflows to provide researchers withaccurate and reliable results. Automated parallel
capillary electrophoresis can analyze multiple samples
at once without researcher intervention. The systems
can house two different gel matrices enabling
unattended and consecutive analysis of multiple RNA
and DNA reagent kits.

Light

Capillaries

Sampleor buffer tray

Charged CoupledDevice (CCD)

Reservoir
High voltage
power supply

Overview of capillary electrophoresis process.

Learn more about the Fragment Analyzer systems

Introduction

QC Instruments

Sample Types

Agilent Femto Pulse System
The Agilent Femto Pulse system delivers
unparalleled sensitivity and sizing of nucleic acids,
enabling accurate and reliable sizing of gDNA
smears and large fragments through 165 kb.

Key benefits

The Femto Pulse system is a powerful and effective
automated, pulsed-field capillary electrophoresis systemwith the ability to separate high-molecular weight(HMW) DNA through 165,000 bp. An optimized opticalplatform allows the system to easily achieve 10 times
higher sensitivity for nucleic acid smears and 100 times
higher for nucleic acid fragments compared to the otherautomated electrophoresis systems. The Femto Pulse wasdesigned for the qualitative and quantitative analysis of
nucleic acid sample with low concentrations or HMW DNA.

Overview of capillary electrophoresis process.

Learn more about the Femto Pulse system

NGS Workflow Solutions

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Agilent 2100 Bioanalyzer System
For more than 20 years, molecular biologists have benefited from RNA and DNA analysis using theAgilent 2100 Bioanalyzer system. Starting in January 2024, the Bioanalyzer instrument will no longer
be available for purchase, however, its corresponding kits and reagents will remain available.
The new generation of Agilent automated electrophoresis instrumentation, including the TapeStation,
Fragment Analyzer and Femto Pulse systems, deliver additional advanced quality metrics for the
analysis of specialized sample types such as genomic DNA, cell-free DNA, and total RNA libraries.

To see how sample analysis using the Bioanalyzer system compares to our other automated electrophoresis instruments,
explore these resources:

01
02
03
04
05
06
07

Comparison of DNA Sample QC for NGS Workflows with the Agilent Fragment Analyzer and Bioanalyzer Systems
Comparison of Small DNA Fragment Analysis using the Agilent Bioanalyzer and Agilent Fragment Analyzer Systems
Comparison of Small RNA Analysis using the Agilent Bioanalyzer and Agilent Fragment Analyzer Systems
Quality Assessment of NGS Libraries using Agilent Automated Electrophoresis Systems
Comparison of the Agilent 2100 Bioanalyzer and the Agilent Fragment Analyzer Systems for Analysis of Plant, Insect, and Bacterial RNA
Comparison of DNA Assays Using the 4200 TapeStation System and 2100 Bioanalyzer System
Performance Characteristics of the RNA and the High Sensitivity RNA ScreenTape Assays for the 4150 TapeStation System

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Proven Quality Metrics for Multiple NGS Applications
Quality metrics provide you with a reliable assessment of sample integrity so you can ensure your nucleic acids are sufficient forsuccessful library preparation and sequencing results. Whether you are working with genomic DNA (gDNA), cell-free DNA (cfDNA),
formalin-fixed paraffin-embedded (FFPE) DNA and RNA, or total RNA you can find reliable quality metrics suited to your application.
Explore each of the metrics below and learn about their use in sample quality control with automated electrophoresis instruments.

Bioanalyzer system

Total RNAFFPE RNAgDNA/
FFPE DNA
cfDNA

RIN

Fragment Analyzer systems

Femto Pulse system

TapeStation systems

RQN

RINe

GQN

DIN

DV200

%cfDNAIntroduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS Quality Control for a Wide Range of Nucleic Acids
Robust sample analysis is crucial throughout the NGS workflow for successful sequencing results. Whether you
need to assess the integrity of your cell-free DNA (cfDNA), formalin-fixed paraffin-embedded (FFPE) DNA and RNA,
genomic DNA (gDNA), total RNA, or NGS libraries, our automated electrophoresis instruments offer you support.
Select your desired sample type to explore relevant application notes and technical overviews:

Genomic DNA
analysis

Total RNA
analysis

FFPE analysis

Cell-free DNA
analysis

NGS library
analysis

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

High Throughput Genomic DNA Assessmentby the Agilent 4200 TapeStation SystemiTechnical overview
Abstract

1,400
1,200

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600

1,000
800
600
400

DIN
1.8

0

900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,00048,500

DIN
5.8

600

DIN
9.8

400

100

250

200

400
250

100

Three different mouse gDNA samples with different DNA integrity
were analyzed using the 4200 TapeStation system. The TapeStation
analysis software displays the results as an electropherogram, a gel
image, and data table. The DIN value is automatically determined, anddirectly displayed under the individual lane of the gel image (A). The
corresponding samples are shown in the electropherogram overlay (B).

[bp]

Sample intensity (FU)When used with the Genomic DNA ScreenTape assay, the 4200
TapeStation system can separate and analyze genomic DNA(gDNA) from 200 to 60,000 bp. It provides an automated numericalassessment of gDNA quality, the DNA integrity number (DIN). The
DIN is calculated using a scale from 1 to 10. A high DIN indicateshighly intact gDNA, and a low DIN suggests a strongly degraded
gDNA sample. The user-independent DIN is the ideal QC tool fornext-generation sequencing (NGS) and array comparative genomic
hybridization (aCGH) workflows.

1,600

Read the full technical overview

Size (bp)Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Evaluating the Agilent 4200 TapeStation System
for High Throughput Sequencing Quality ControliApplication Note
Abstract

The bar chart illustrates that the DNA integrity analysis with the 4200 TapeStation system
is highly comparable to that of the 2200 TapeStation system. The precision for the DNA
integrity analysis for both systems is 4% for all tested samples and concentrations.

[bp]

gDNA sample A
no dil. 1:2
1:4

gDNA sample B
no dil. 1:2
1:4

Agilent 2200 TapeStation system[bp]

gDNA sample A
no dil. 1:2
1:4

gDNA sample B
no dil. 1:2
1:4

DIN
9.4

DIN
8.8

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400
250

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400
250
100

100

DIN
9.1

DIN
9.5

DIN
9.5

DIN
8.7

DIN
8.6

DIN
9.4

10

DIN
9.5

DIN
9.7

8
7
6
5
4
3
2
1
0

DIN
8.6

Agilent 4200
TapeStation system
Agilent 2200
TapeStation system

9DIN

To evaluate the difference of the DNA integrity number (DIN) between the 2200 and the4200 TapeStation systems, two commercially available gDNA samples were analyzed
at three different concentrations using the Genomic DNA ScreenTape assay. The DNA
integrity number (DIN) is automatically determined and directly displayed below theindividual lane of the gel image. A DIN is calculated on a scale from 1 to 10. A high DIN
indicates highly intact gDNA, whereas a low DIN corresponds to a strongly degraded
gDNA. Both gDNA samples were highly intact, as indicated by the determined DIN, which
is displayed below the gel image.

Agilent 4200 TapeStation system

no dil.
1:2
1:4
gDNA sample A

no dil.
1:2
1:4
gDNA sample B

Read the full application note

DIN
9.6

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Quality Control for Agilent SureSelect QXTWGS Library PreparationiApplication note
Abstract
[bp]

70

B1

C1

DIN
9.2

DIN
7.9

DIN
8.3

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400

50
40
30
20
10
0

A1

Qubit
Agilent 4200 TapeStation
Nanodrop60

Concentration (ng/µL)

The Agilent SureSelect QXT WGS protocol requires high-quality DNA samples
for optimal performance and precise quantification of the gDNA starting
material. Serial quantification was carried out using the Qubit instrument andthe dsDNA BR assay in accordance with the protocol. The same samples wereanalyzed on the 4200 TapeStation system with the Genomic DNA ScreenTape
assay and the NanoDrop with six replicates on each instrument. Data fromthe 4200 TapeStation system, Qubit, and NanoDrop are presented in the
figure, showing the applicability of the Genomic DNA ScreenTape assay inquantitating genomic DNA starting material. The quantification results of the
TapeStation system correlate with the Qubit instrument. The measurement ofgenomic DNA with UV spectroscopy tends to overestimate the quantity due toother buffer components that may absorb in the UV spectrum.

250
First dilution

Second dilution

In addition, the Genomic DNA ScreenTape assay provides an objective
assessment of sample integrity within the same QC step. Sample integrity
is automatically determined by the DNA integrity number (DIN) calculation
provided by the TapeStation analysis software.
Read the full application note100Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

GQN Quality Metrics with the Fragment
Analyzer and Femto Pulse SystemsiApplication note
16,00014,00012,00010,000
8,000
6,000
4,000

B

Average smear size (bp)12,147
23,33945,30457,78973,267
94,045
109,968
164,292

GQN Set at 30 kb
0
1.5
6.4
7.1
7.8
7.8
8.2
8.8

Read the full application note

165,500

42,00050,00017,70023,0000

10,000

2,000
1,300

The GQN is commonly used for evaluating the input gDNA material for
NGS library preparation. To prepare a successful library, the sample
must be of the correct size and of sufficient quality for sequencing.
The GQN threshold can be set by the user to reflect the size thresholdnecessary for their particular requirements. The GQN is given on ascale of 0 to 10, with a higher score indicating that more of the sample
exceeds the user-defined threshold. In this example, the Femto Pulse
system was used to report the average smear size (A) and GQN
set at 30 kb (B) of several gDNA samples that had been sheared tovarious sizes. For NGS, quality metrics can aid decisions about library
preparation, including which samples to use for input material, thenumber of PCR cycles, and the amount of library to use for enrichment.

LM

1

Quality assessment of nucleic acids is critical to the success of many
downstream applications, including next-generation sequencing (NGS).
The Fragment Analyzer and Femto Pulse systems provide quick andeasy assessment of genomic DNA (gDNA) quality and integrity with the
genomic quality number (GQN).

18,000 A

RFU

Abstract

Size (bp)Introduction

A

i

6,000
5,000
4,000

LM

2,000
1,000

1,000

48,500

6,000
10,00015,000

3,000

1,500

800

1,000

600

400

200

0
Size (bp)

Read the full application note

6,000
10,00015,000

3,000

1,500

800

1,000

600

400

48,50048,500

2,000

LM

6,000
10,00015,000RFU

3,000

Size (bp)

Cotton (short)
Cotton (ultrashort)
Coriell (short)
Coriell (ultrashort)E. coli (short)E. coli (ultrashort)

DNF-468

3,000

LM

4,000

D

1,500

DNF-467

6,000
5,500
5,000
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0

1,000

Cotton (short)
Cotton (ultrashort)
Coriell (short)
Coriell (ultrashort)E. coli (short)E. coli (ultrashort)

800

C

Size (bp)200

48,500

6,000
10,00015,000

3,000

1,500

800

1,000

600

400

0

1
75

RFU

7,000

3,000

6,000
5,000

Cotton (DNF-467)
Cotton (DNF-468)
Coriell (DNF-467)
Coriell (DNF-468)
E. coli (DNF-467)
E. coli (DNF-468)

Ultrashort array

600

7,000

NGS Workflow Solutions

B

400

8,000

8,000

RFU

LM

1
75

Abstract
The quality and concentration of genomic DNA (gDNA) starting
material is crucial for successful downstream long-read and wholegenome sequencing. Quality analysis for gDNA with varying rangesof concentrations can be performed on the Fragment Analyzer
systems with the Genomic DNA 50 kb kit and the HS Genomic DNA
50 kb kit. The Genomic DNA 50 kb kit offers a concentration rangeof 25 to 250 ng/μL input gDNA, while the HS Genomic DNA 50 kb kit
has a lower concentration range of 0.3 to 12 ng/μL input gDNA for
low concentrated samples. Genomic DNA from cotton, E. coli, andhuman (Coriell) were compared on both kits with the FA 12-Capillary
Array Short, 33 cm (short array), and FA 12-Capillary Array Ultrashort,
22 cm (ultrashort array). On both kits, the short and ultrashort arrays
demonstrated consistent sizing for the three sample types. The
ultrashort array offers the convenience of shortened run times whileproviding comparable gDNA sizing, concentration, and genomic quality
number (GQN) compared to the short array with both kits.

Short array

200

RFU

Assessment of Genomic DNA Quality with
the Agilent 5200 Fragment Analyzer System
Application note

9,000

Cotton (DNF-467)
Cotton (DNF-468)
Coriell (DNF-467)
Coriell (DNF-468)
E. coli (DNF-467)
E. coli (DNF-468)

1
75

6,000
5,500
5,000
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0

Sample Types

1
75
200

Genomic DNA analysis

QC Instruments

Size (bp)Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Genomic DNA Sizing and Quality Control
on the Agilent Femto Pulse SystemiApplication noteThis application note shows comparable sizing of four gDNA
samples separated on the Femto Pulse and traditional PFGE. It also
demonstrates consistent sizing of samples throughout a dilution
series and discusses the application of genomic quality number (GQN)
provided by the Femto Pulse system.

A

9,000

LM
179,342 bp

8,000
RFU

7,000
6,000

5,600

B

165,500

50,00042,00017,70021,00023,000

1,300

1

4,000

10,0005,000
Size (bp)

LM

5,400
5,200

157,440 bp

5,000
4,800
4,600
4,400

Read the full application note

165,500

50,00042,00017,70021,00023,00010,000

4,000

1,300

4,200
1

Large-insert library preparation relies upon multiple quality control
steps. Typically, overnight pulsed-field gel electrophoresis (PFGE)
separations are used to assess gDNA over 50 kb in size. The Agilent
Femto Pulse system is the only instrument on the market capable ofreplacing PFGE with fast, automated assessment of high-molecularweight (HMW) gDNA, saving time and money in the preparation of largeinsert libraries. The Genomic DNA 165 kb kit offers two pulsed-field
capillary electrophoresis separation methods. The gDNA 165 kb method
is a 70-minute method recommended for gDNA under 80 kb. The
extended Genomic DNA 165 kb method provides enhanced separationand sizing for larger samples in 3.5 hours. Large gDNA separated withthe fast method displayed a sharp, compact peak around 165 kb (A).
The same sample analyzed with the extended method resulted in a
broader smear representing the entire sizing range of the sample (B).

10,000

RFU

Abstract

Size (bp)Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Comparison of Different Methods to Isolate HMW
DNA from Bacteria for Nanopore SequencingiApplication note
Abstract

35000

The first step in many sequencing experiments is to isolate nucleic acids
from a specimen. As long-read sequencing technologies have advanced,
many commercial companies now offer kits specific to the extraction of highmolecular weight (HMW) genomic DNA (gDNA). However, the size and qualityof the gDNA can vary greatly depending upon the extraction method used. For
sequencing facilities, this can affect the type of sequencing that is performed,
making knowledge of sample integrity crucial to a successful experiment.

25000

LM

32500

RFU

30000
27500
22500
20000
17500
17500
15000
10000
7500

165500

42000
50000

1300

1

1857

10000
17700
21000
23000

5000

Size (bp)# of reads (logarithmic scale)

B
0.9

0.6

0.3

0.0
1,000

10,000100,000

Fragment length (bp)C
# of reads sequenced

In this application note, researchers at the NGS Competence Center
Tuebingen (NCCT) used the Agilent Femto Pulse system to analyze HMW
gDNA from five different commercially available extraction kits. The sizeof the input material was then compared to the sequenced read lengths todemonstrate the impact that the isolation method can have on sample sizeand sequencing results. An example from one of the five extraction kits isshown in the figure. Data generated from the Femto Pulse demonstrated thatthe extraction method used resulted in a large peak with some smearing to
the left of the peak, indicating the presence of smaller fragments within thesample (A). The distribution of the sample was confirmed with Nanopore
sequencing results, which showed a large amount of fragments of smaller
sizes (B), but also some as big as 500 kb (C).

A

136625

39201
37500

9e–05
6e–05
3e–05
0e–00
100 kb200 kb300 kb400 kb500 kbRead length

100 kb200 kb300 kbRead the full application note

400 kb500 kb

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Comparison of Agilent Femto Pulse System
Sizing with Long-Read Sequencing Read LengthiApplication note
15 kb BP

11470
11000
10500
10000
9500
9000
8500
8000
7500
7000
6500
6000
5500
5000
4450

LM

Weighted Histogram of read lengths

Size (bp)Weighted Histogram of read lengths

D

Number of bases

Number of bases

40000000

30000000

20000000

10000000

20000

40000

60000

80000

Read length

1000000020000 40000 60000 80000 100000 120000 140000

Read length

Average size of input gDNA compared
to mean sequencing read length
25000

(bp)

20000
15000

165500

42000
50000

10000
17700
21000
23000

1300

1

RFU

Size (bp)165500

42000
50000

1300

10000
17700
21000
23000

RFU

Long-read sequencing results can be maximized by loading only
long fragments onto the sequencer, thereby eliminating any issues
with preferential sequencing of smaller fragments. This can beachieved through size selection, to exclude the portion of the sample
4762
below a specified threshold. Sheared samples, with and without size
selection, were analyzed with the Agilent Femto Pulse system, andthen sequenced on the Oxford Nanopore Technologies MinION. The
size distribution of the samples reported by the Femto Pulse was
40000000
similar to the histogram of the sequencing read lengths. Shown here
35000000is an example of a 15 kb Blue Pippin (15 kb BP) size-selected gDNA
30000000
analyzed on the Femto Pulse (B). The sample was sequenced with
25000000the Oxford Nanopore Technologies MinION, and histograms of the20000000
read lengths were generated from the data with NanoPlot (D). Size15000000
selection eliminated the small fragments present in the sample, 10000000
resulting in similar sizes between the Femto Pulse average size and5000000
the mean sequencing results. The Femto Pulse confirmed effective 0 0
size selection, which was further confirmed by the sequencing results.

LM

9746

10586
10000
9500
9000
8500
8000
7500
7000
6500
6000
5500

12743

B

20 kb g-TUBE sheared

1

Abstract

Read the full application note

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Comparison of Constant- and Pulsed-Field
Electrophoresis Technologies for Analysis of High
Molecular Weight and Large DNA Fragments
Application note

A

i
C
38000
36000

Abstract

32000
30000
28000
26000
24000
22000
Size (bp)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-fieldtechnology is traditionally thought to be best suited for separation of DNA
smears that are less than 20 kb, based on previous studies. Beyond 20 kb, DNAsmears 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 thesedifferent technologies: the Agilent Fragment Analyzer systems use constantfield technology, while the Agilent Femto Pulse system uses both pulsed- andconstant-field technologies. As shown in the figures, the data presented in thisapplication note indicates that while both the Fragment Analyzer and Femto
Pulse can be used for analysis, the Femto Pulse provides more accurate sizing
for high molecular weight (HMW) DNA, genomic DNA (gDNA), and sheared
samples, both within the range of 10 to 20 kb, and above.

34000

20000
18000
16000
14000
12000

B

10000
8000
6000
4000
2000
0

Sample A

Sample B

Fragment Analyzer gDNA 50 kb kit

Sample C
Femto Pulse gDNA 165 kb kit

Expected sample sizeA) 10 kbB) 15 kbC) 20 kb

Read the full application note

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Genomic DNA analysis

Impact of gDNA Integrity on the
Outcome of DNA Methylation StudiesiApplication note
Abstract

5.4
5.4

1.0
1.0

DIN
DIN5.85.8
5.45.4

4.8
4.8

3.6
3.6

0.6
0.6

log (N)
log1010(N)DIN 8.0
8.0
DIN

3.0
3.0
2.4
2.4

0.4
0.4

1.8
1.8
1.2
1.2

0.2
0.2

4.84.80.8
0.8

4.24.20.6
0.6

3.63.60.4
0.4

2.42.4log (N)
log1010(N)

4.2
4.2

DIN 8.0
DIN 8.0

0.8
0.8

3.03.01.81.80.2
0.2

1.21.2

0.6
0.6
00
00

1.0
1.0

0.2
0.2

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4.2
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log (N)
log1010(N)

3.0
3.0

4.04.00.6
0.6

DIN 8.0
DIN 8.0

3.6
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0.4

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1.8
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DIN7.1
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00

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DIN7.3
7.3

5.4
5.4

log (N)
log1010(N)This application note focuses on 5-methylcytosine (5-mC) DNA methylationand its detection. By far, the most commonly used method for DNA
methylation analysis is bisulfite sequencing due to its high-resolution
detection when combined with sequencing. Advances in next-generation
sequencing made it possible to perform bisulfite sequencing at a genomewide scale. The DNA methylation analysis based on reduced representation
bisulfite sequencing, a high-throughput technique, allows analysis on asingle nucleotide level. This method is based on the enrichment of genome
regions with high CpG content (sites within the genome where a cytosine is
next to a guanine) using a combination of restriction enzymes and bisulfite
sequencing and is applicable to any species with a reference genome.
Shown here is a comparison of the DNA methylation levels for overlapping
CgP sites of four FFPE samples. DIN values ranged from 4 to 7.3, to a fresh
frozen sample with a DIN value of 8 using Hexbin plots.

DIN
DIN4.0
4.0

DIN8.08.0
DIN

DNA methylation is the most widely studied modification involved in
epigenetics. In mammalian cells, DNA methylation mainly involves thetransfer of a methyl group from S-adenosyl methionine to the carbon
5 position of a cytosine residue to produce 5-methylcytosine. DNA
methylation is mainly implicated in the repression of transcriptional activity.

1.0
1.0

00
00

0.2
0.2

Read the full application note

0.4
0.6
0.4
0.6
DIN
DIN7.3
7.3

0.8
0.8

1.01.000

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Comparison of Small RNA Analysis
using the Agilent Bioanalyzer andAgilent Fragment Analyzer
Technical overview

iA

Abstract
Analysis of small RNA samples can help with optimization of small RNA
isolation and purification protocols and ensure successful downstream
applications such as small RNA next-generation sequencing (NGS) (RNASeq), miRNA microarrays, and qRT-PCR. The systems of the automatedelectrophoresis portfolio from Agilent have been well established for the
analysis of total RNA, and uniquely offer quality analysis of small RNAs
with either the Agilent 2100 Bioanalyzer system, or the Agilent Fragment
Analyzer systems. Both instruments use kits that focus on small RNA andmiRNA quality control, concentrating on a narrow range of 200 nt and below,
and allowing for high-separation resolution of small RNAs. In this technicaloverview, samples are compared across both the Bioanalyzer and the
Fragment Analyzer Small RNA kits to demonstrate the equivalency of thesystems for analyzing small RNAs.The figure shows a commercially available
total RNA that was further analyzed on A) the Agilent Bioanalyzer Small RNA
kit and B) the Agilent Fragment Analyzer Small RNA kit to allow for higher
separation resolution of the small RNA portion of the sample below 200 nt.

BRead the full technical overview

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Evaluating the Agilent 4200 TapeStation
System for High Throughput Sequencing
Quality Control
Application note

i

A
×103
16

Abstract

9
8

10

7

7

RINe

6

6

Liver total RNA
1:8
1:20

Marker

4

5
4
3

1:35

2

2

1
4,000
6,000

2,000

1,000

0
500

0

200

0

Agilent RNA ScreenTape assay
10

12

25

The RNA integrity number equivalent (RIN ), indicating RNA integrity,
is automatically determined by the TapeStation software. The RINeis calculated on a scale from 1 to 10. A high RINe indicates highly
intact total RNA, whereas a low RINe corresponds to a strongly
degraded sample. The bar chart (B) illustrates that RNA integrity
analysis is highly reproducible and independent of the analyzed
concentration for both TapeStation systems. In comparison to the2200 TapeStation system, the 4200 TapeStation system further
reduces manual operating time, enabling the analysis of 96 samples
without physical intervention. This is a primary requirement for highthroughput NGS labs.
eSample intensity (FU)As RNA is highly sensitive to degradation, quality control is essential,
and usually includes RNA integrity analysis and quantification. The
electropherogram overlay shown here represents different dilutionsof liver total RNA analyzed on the 4200 TapeStation system with the
RNA ScreenTape assay (A).

B

14Size [nt]

1:8

1:20

1:35

Agilent 4200 TapeStation system
Agilent 2200 TapeStation system

Read the full application note

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Quality Control in Illumina Sequencing
Workflows Using the TapeStation SystemiApplication note

18
S
28
S

w
Lo

0

Read the full application note

6,000

Size
(bp)

2,000

6,000

2,000

500

1,000

200

25

0

1

1,000

1

2

500

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25

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er

B
×103
5

Sample intensity (normalized FU)

28
S

w
Lo

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S

A
×103
5

Sample intensity (normalized FU)

RNA is sensitive to degradation due to the ubiquitous presence of
RNase and its more fragile single-stranded structure. Therefore,monitoring the integrity of starting material is indispensable, andprocessing a reference sample as positive control throughout thelibrary preparation and sequencing is highly advisable. With the
RNA ScreenTape assay, the RNA integrity number equivalent (RINe)delivers an objective assessment of the integrity of RNA starting
material. RINe is a proven equivalent to the widely accepted quality
metric RIN. The fragmentation conditions of RNA‑seq protocols usedby the sequencing core facility are optimized for high-quality RNA;
more precisely, a RINe of 8.0 or higher is recommended for successful
library preparation. The figure shows two samples close to thisthreshold, one passing (A) and one failing (B) the quality requirement.
The use of degraded RNA can result in low yield, over-representationof 3’ ends of the RNA molecules, or failure of the protocol.

er

Abstract

Size
(bp)

A
A 1,400
1,400
1,200
1,200

Total RNA analysis

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

LM
LM

1,000
1,000

RFU
RFU

Quality Analysis of Eukaryotic Total RNA with
the Agilent 5200 Fragment Analyzer System

i

800
800
600
600
400
400

6,000
6,000

3,000
3,000

2,000
2,000

1,500
1,500

1,000
1,000

500
500

4,000
4,000

The RNA quality number (RQN) is a user-independent quality
metric for easy evaluation of total RNA quality. Total RNA quality
is a constant concern because of how easily RNA degrades
due to heat, RNase exposure, and improper handling. The 5200
Fragment Analyzer system and RQN metric were used to analyze
universal mouse reference total RNA degradation over time.
Electropherogram (A) and digital gel images (B) allow for the
total RNA profiles to be compared at different time points of heatdegradation. As shown, the 18S ribosomal peak becomes smaller,
while the 28S ribosomal peak completely disappears as the sample
is further degraded. This degradation strongly correlates with adecrease in RQN (C), allowing for easy assessment of RNA quality.

Size (nt)
Size
(nt)

F10

F11

F12

A
B

Size
Size (nt)
(nt)
6,000
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C

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Minutes at 70 °C

14

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RQN

Abstract

00

200
200

Application note

15
15

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200

6
5
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0

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16

Read the full application note18

20

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Performance Characteristics of the RNA andHigh Sensitivity RNA ScreenTape Assays forthe 4150 TapeStation System
Technical overviewiA [nt]

A1

B1

C1

D1

E1

F1

G1

H1

A2

B2

C2

D2

RINe
3.2

RINe
3.1

RINe
3.2

RINe
5.0

RINe
5.0

RINe
5.0

RINe
6.7

RINe
6.8

RINe
6.9

RINe
8.6

RINe
8.6

RINe
8.5

Abstract
2,000
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25

×103

Sample 4

B

30

Sample 3
Sample 225

Sample 1

20

15

10

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6,000

2,000

1,000

500

0

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25

Four rat kidney RNA samples with different degradation stages were analyzed using
the 4150 TapeStation system. The 4150 TapeStation software displays the results
as an electropherogram, a gel image, and data table. The RINe value is automaticallydetermined, and directly displayed under the individual lane of the gel image (A). (B)shows the corresponding electropherogram overlay. Comparison of the RINe from the4150 and 4200 TapeStation systems was evaluated and found to be equivalent.

6,000
4,000

Sample intensity (normalized FU)

RNA serves as input material within various gene expression analysis techniques
like RNA-Seq, microarray, and RT-qPCR. RNA is very sensitive to degradation andits integrity critically affects the success of the downstream applications. Quality
control of RNA input material is crucial for ensuring high-quality results. The 4150
TapeStation system can be used with the RNA ScreenTape assay to separate andanalyze RNA, providing an automated numerical assessment of RNA quality, the RNA
integrity number equivalent (RINe). The RINe is calculated using a scale from 1 to 10.
A high RINe indicates highly intact RNA, and a low RINe suggests a strongly degraded
RNA sample. The RINe was demonstrated to be equivalent to RIN from the 2100
Bioanalyzer system. The user-independent RINe is the ideal QC tool for next-generation
sequencing (NGS) workflows.

Read the full technical overview

Size (nt)Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Monitoring Library Preparation for
Next-Generation Sequencing in
Systems Biology Omics Analysisi

Application note

The same samples were quantified fluorometrically and results werecompared with the RNA ScreenTape assay. Both assays resulted ina similar total RNA concentration of samples, with a slope of 1.017
and a R2 of 97.9%, showing high correlation (B). Two degraded RNA
samples were treated as outliers. Overall, the RNA ScreenTape assay
provides a very useful tool in determining integrity and quantity ofRNA starting material for sequencing in a single analysis step.

B

800

10

Concentration (ng/µL) RNA ScreenTape assay

RNA samples are often subject to degradation by RNases, chemical,
or other environmental impacts. Therefore, RNA integrity analysis
is a crucial step before any downstream application like NGS. The
RINe metric from the TapeStation software is equivalent to the RIN
metric provided by the RNA assays of the Bioanalyzer system. The
comparative analysis of 30 RNA samples extracted from mousecancer tissue verified that RIN and RINe highly correlate, with a slopeof 0.997 and a goodness-of-fit (R2) of 97.9% (A).

A

9
8

RINe (4150 TapeStation system)

Abstracty = 0.9973x – 0.1922
R² = 0.9788

7
6
5
4
3
2

600
500
400
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1
0

y = 1.0172x + 16.555
R² = 0.9791

700

0

1

2

3
4
5
6
7
RIN (2100 Bioanalyzer system)

8

9

10

0

0

100

Read the full application note

200

300

400

500

Concentration (ng/µL) Qubit RNA HS assay

600

700

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Comparison of RIN and RQN for the
Agilent 2100 Bioanalyzer and the
Fragment Analyzer SystemsiTechnical overview
SS RNA Kits
Eukaryotic RNA
12.0
10.0
Bioanalyzer

The Bioanalyzer instrument is well established for providing a reliable,
automated RNA integrity number (RIN). The RIN provides an objective
assessment of RNA integrity. The Fragment Analyzer offers a userindependent quality metric, the RNA quality number (RQN), for easy
evaluation of total RNA quality. Both the RIN and RQN consider theentire electropherogram, with scoring from 1 to 10, where 10 indicatesthe highest possible RNA quality and 1 completely degraded RNA.
Eukaryotic samples with a varying degree of RNA integrity, fromcompletely intact, to mildly and strongly degraded, were compared on
the Bioanalyzer and Fragment Analyzer instruments. Both the standard
sensitivity (A) and High Sensitivity RNA (B) kits on both instruments
provided comparable RIN and RQN scores throughout the degradation
series. This is demonstrated with the slope and R2 value close to 1.
This technical overview also shows a strong correlation between the
RIN and RQN for prokaryotic E. coli RNA samples.

A

8.0
6.0

y = 0.9574x + 0.4957
R² = 0.9628

4.0
2.0
0.0
0

2

4

6

8

10

12

Fragment Analyzer

B

HS RNA Kits
Eukaryotic RNA
1210
Bioanalyzer

Abstract86

y = 0.9875x + 0.0423
R 2 = 0.9746

4
2
0
0

2

4

6

8

Fragment Analyzer

Read the full technical overview

1012Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis
A) Agilent Bioanalyzer System

Comparison of RNA Quality Analysis with the
Qubit RNA IQ Assay and Agilent AutomatedElectrophoresis Systems

10
5
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[nt]
Black: intact
Red: mildly degraded
Blue: severely degraded

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Red: intact
Blue: mildly degradedGreen: severely degraded[FU]

Size (nt)

C) Comparison of quality scores

1050Human Heart RNA Quality Scores
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[nt]

4000

B) Agilent Fragment Analyzer System

Black: intact
Red: mildly degraded
Blue: severely degraded

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Human heart intact

15

RFU

500

B) Agilent Fragment Analyzer System

A) Agilent Bioanalyzer System

Size (nt)

C) Comparison of quality scores
Human Heart RNA Quality Scores
10.0

Read the full technical overview

8.0
6.0
4.0
2.0
0.0

200

3000
4000

25

Abstract

-1500

i

40
35
30
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Technical overview

RNA integrity can be determined using electrophoresis methods that
separate the sample based on size, allowing for the distinction of theribosomal peaks, small RNA, and any degradation products. The Agilent
2100 Bioanalyzer system and the Agilent Fragment Analyzer systems areused for RNA quality control (QC), providing information about both thequantity and the quality of a given sample. The systems both generate an
objective, reliable quality metric score for each sample analyzed. Thesescores are known as the RNA integrity number (RIN) for the Bioanalyzer,
and the RNA quality number (RQN) for the Fragment Analyzers. Eachscore is based on a scale from 1 to 10, with 1 identifying significantly
degraded samples and 10 identifying high-quality, intact RNA specimens.
The RNA Integrity and Quality (IQ) Assay kit for the Qubit 4.0 fluorometerfrom Thermo Fisher Scientific also offers an RNA quality score,
representative of the ratio of small and large RNAs present in the sample.
In this technical overview, the RIN and RQN scores from the Agilent
automated electrophoresis instruments are compared to the Qubit RNA IQ
score across a series of RNA reference samples from multiple species.

Red: intact
Blue: mildly degradedGreen: severely degraded[FU]

RIN (Bioanalyzer)

RQN (Fragment Analyzer)

RNA IQ (Qubit)

RQN (Fragment Analyzer)
Human heart mild degradation

RNA IQ (Qubit)

Human heart severe degradation

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Total RNA analysis

Comparison of the Agilent 2100 Bioanalyzer
and the Agilent Fragment Analyzer Systems
for Analysis of Plant, Insect, and Bacterial RNA

iTechnical overview
Abstract
A
B

[FU]
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11335
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Size (nt)

Read the full technical overview

4000

3000

2000

1500

1000

500

1500
15

40

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60

RFU

Evaluating RNA integrity prior to downstream analysis saves time, effort,
and resources by ensuring that only samples of sufficient quality are used.
To aid in objectively determining the integrity of a sample, quality metrics
can be assigned that help to grade the quality of the RNA, independentof user bias. The Agilent 2100 Bioanalyzer and 5200 Fragment Analyzer
systems each provide reliable, convenient analysis of samples. Eachsystem also provides a quality metric for RNA samples, called the RNA
integrity number (RIN) and the RNA quality number (RQN), respectively.
The RIN and RQN assign a score from 1 to 10 for a given RNA sample. A
score of 1 indicates severely degraded RNA, while a score of 10 indicateshighly intact RNA. In this technical overview, different total RNA samples
were evaluated to compare the quality metrics between the Bioanalyzer
and Fragment Analyzer systems. Plant, insect, and bacterial total RNA
samples were analyzed using both the high- and standard sensitivity kits
for each system, to provide a detailed comparison of the RIN and RQN.
The electropherograms here show corn analyzed on the (A) Agilent 2100
Bioanalyzer system and (B) Agilent 5200 Fragment Analyzer system.

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

FFPE analysis

Use of the Agilent 4200 TapeStation System
for Sample Quality Control in the
Whole Exome Sequencing Workflow at theGerman Cancer Research Center (DKFZ)iApplication note
Abstract
F4

G4 H4 A5 B5 C5 D5 E5

F5

G5 H5

400

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400

300

200

100

250

Read the full application note

900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,00048,500600

DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN
2.9 4.9 3.9 3.1 2.6 1.5 4.4 4.4 3.2 3.6 2.5 2.3 3.3 3.7 3.5

400

0

100

250

Both figures show a representative subset of samples analyzed on
the 4200 TapeStation with the Genomic DNA ScreenTape assay.
gDNA samples extracted from FPPE material often have low
DNA integrity but can still be sufficiently intact for whole-exome
sequencing library preparation protocols and successful sequencing.

B
B4 C4 D4 E4100This initial QC includes quantification and analysis with a4200 TapeStation system and the Genomic DNA assay to determine
DNA quality, based on the DNA integrity number (DIN).

A[bp]

Sample intensity [FU]To determine if samples were suitable for NGS library preparation,a quality control (QC) assessment was performed at the beginning
for a batch of 88 genomic DNA (gDNA) samples from FFPE
tumor tissue by the German Cancer Research Center (DKFZ) HighThroughput Sequencing Unit.

500

Size (bp)Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

FFPE analysis

The DNA Integrity Number (DIN) Provided by
the Genomic DNA ScreenTape Assay Allows for
Streamlining of NGS on FFPE Tissue Samples

iApplication note
Abstract

bp

1

2

3

4

5

6

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400
48,50015,000
7,000
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7,000
15,00048,500

400

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0
100

Sample intensity (FU)

Sequencing of genomic DNA (gDNA) from FFPE archived tissue can bechallenging, as the obtained material is often of variable quality. This study
demonstrates that the DNA integrity number (DIN) obtained by the quality
control of gDNA using the Agilent Genomic DNA ScreenTape assay has
allowed for a pronounced saving of sequencing and sample preparation
overhead. Out of a total of 751 FFPE samples, a subset of 197 were tested
for a correlation of various NGS parameters against the DIN. A correlation
was identified between DIN and the key parameters of on-target rateand coverage at 10x. A QC threshold of ≥ 3 DIN was therefore set, whichconsequently excluded 65% (n = 488) of the total sample set and saved asignificant amount of time and effort. The right panel is the gel image, andthe left panel is the electropherogram.

600

Size (bp)

DIN DIN DIN DIN DIN DIN
4.1 5.2 5.0 2.2 2.9 1.9Read the full application note

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

FFPE analysis

FFPE Sample Quality for the MGISEQ-2000
Sequencing Platform with the Agilent
TapeStation SystemiApplication noteNo.

Concentration(ng/µL)

Main Peak(bp)

FFPE-1

Cervical

2.9

28.6

1,919

FFPE-2

Colorectal

4.5

84.8

2,241
731

FFPE-3Thyroid

2.6

42.4

FFPE-4

Breast

2.2

10.2

574

ControlStandards

7.9

32.4

24,312

A1) gDNA kit ladder (100 bp - 48.5 kb)
B1 to E1) FFPE samples 1 to 4
H1) FFPE DNA standard

1200

Composite
A1 B1 C1 D1 E1 H1

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Formalin-fixed paraffin embedding (FFPE) is one of the morecommon ways to preserve clinical samples, and FFPE tumor samples
serve as valuable study materials for clinical and translational
medicine research. To ensure successful and reliable sequencing of
FFPE-derived DNA, it is necessary to conduct quality control (QC) of
both the initial DNA, as shown here, as well as fragmented samples
at key points during next-generation sequencing (NGS) library
construction. In this application note, the Agilent 4150 TapeStation
system was used to conduct QC of the entire library construction
process for five FFPE DNA samples used on the MGI Tech MGISEQ2000RS sequencing platform. High-quality sequencing results wereachieved by stepwise QC throughout the library preparation process,including the initial sample, pre- and post-PCR steps, and the final
library, helping to ensure high-quality sequencing.

DIN

Sample Intensity [Normalized FU]

Abstract

Type of Tissue

Read the full application note

Size
[bp]

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

FFPE analysis

DV200 Evaluation with RNA ScreenTape AssaysiTechnical overview
Abstract
The DV200 quality metric represents the percentage of RNA
fragments above 200 nucleotides and shows a high correlation
to the precapture library yield of RNA samples originating fromformalin-fixed paraffin-embedded (FFPE) tissue. The RNA and HighSensitivity RNA ScreenTape assays enable fast and easy analysis of
FFPE RNA samples, with the TapeStation software displaying DV200
results after region setup as percentage of total. DV200 region setup
can be automated for repeated FFPE RNA sample analysis and all
region data can be exported and reported. Both the RNA ScreenTape
assay and High Sensitivity RNA ScreenTape assay yielded highly
comparable results.

Read the full technical overview

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Cell-free DNA analysis

Detection of Contaminating High Molecular
Weight DNA with the Cell-Free DNA
ScreenTape Assay
Technical overview

iA. Agilent 2100 Bioanalyzer system
[FU]

UM

160

Abstract

LM

120

Mononucleosome

100
80

Dinucleosome

60
40

HMW region

Trinucleosome

20

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er

B. Agilent 4200 TapeStation system
0
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Mononucleosome

400

300

Dinucleosome

HMW DNA

200

Trinucleosome

100

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Sample Intensity [Normalized FU]

Cell-free DNA represents a challenge for next-generation sequencing (NGS)
workflows due to the low yield, complex fragmentation pattern, and possibilityof contaminating high molecular weight (HMW) DNA. To appropriately assess
the quality of cfDNA, it is important to visualize the fragmentation pattern
and any degradation or contamination within the sample. Assessment of the
cfDNA fragments can be performed using Agilent automated electrophoresis
instruments, including the Bioanalyzer and TapeStation systems, as shownin the figure. However, the presence of HMW DNA contamination, which canlead to misrepresentation of the total sample concentration and negatively
affect NGS library yield and sequencing results, can overlap with the Upper
Marker used by the Bioanalyzer, impacting assessment of the total sample.
Alternately, the Cell-free DNA ScreenTape assay for the TapeStation systems
reliably visualizes HMW DNA contamination and calculates a %cfDNA
quality score for objective assessment of the amount of cfDNA in thesample compared to any HMW DNA contamination. This technical overview
compares the analysis of cfDNA contaminated with HMW DNA between the
Bioanalyzer and TapeStation systems.

140

Read the full technical overview

Size
[bp]

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Cell-free DNA analysis

Performance Characteristics of the Agilent
Cell-free DNA ScreenTape Assayi

Technical overview
Abstract

2000 pg/µL
1500

800 pg/µL
400 pg/µL
200 pg/µL
80 pg/µL

1000

700

600

500

400

300

200

150

100

75

50

0

Read the full technical overview

1000

500

35

Sample Intensity [Normalized FU]

To demonstrate the concentration independence of the %cfDNA
metric, a dilution series of a reference cfDNA sample (n=24),
covering the entire concentration range of the assay, was analyzed.
The average %cfDNA value was 85.0% ± 1.0 with a minimum valueof 83.7% and maximum value of 86.6%. The %cfDNA results andthe electropherogram profiles were consistent over the entire
concentration range of the assay. In the figure, each concentration
is overlaid with three replicates to demonstrate the consistency
and precision of the Cell-free DNA ScreenTape assay. These results
show that the %cfDNA quality metric provided by the Cell-free DNA
ScreenTape assay is highly accurate and precise, and that thepercentage is independent of the sample concentration.

4500 pg/µL

Size
(bp)

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Cell-free DNA analysis

cfDNA Separated on the
Agilent Femto Pulse SystemiApplication note
250 pg/µL
125 pg/µL
62.5 pg/µL
31.3 pg/µL
15.6 pg/µL
7.8 pg/µL

LM

1,600
1,400
1,200
RFU

UM

1,000
800
600
400

B
1,400

1,170
963

1,000
783

800
574

600
375

400

0

174

1

2

3
4
cfDNA fragments

5

6

Read the full application note

6,000

3,000

2,000

1,500

1,200

1,000

900

800

700

Sizing

1,200

200

600

500

400

100

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0

200

200
1

The Femto Pulse system offers unparalleled sensitivity for the
analysis of low concentrated cfDNA samples. In addition, the system’shigh resolution provided allows samples with multiple fragments tohave complete separation between peaks. cfDNA samples can differ
in the number of fragments present, but the mono- and dinucleosome
fragments are usually both present. cfDNA analyzed on the Femto
Pulse with the Ultra Sensitivity NGS kit displayed six fragments (A). In
the dilution series (250 to 7.8 pg/μL), the first three cfDNA fragments
were completely separated at all concentrations, with the fourthfragment easily distinguishable down to 15.6 pg/μL. The fifth andsixth fragment peaks were less apparent below 31.3 pg/μL. A seventh
fragment peak was observed only in the highly concentrated samples,62.5 pg/μL and higher. Sizing remained consistent for all six peaks
throughout the concentration range they were visible for (B).

1,800

Size (bp)

AbstractA

Size (bp)Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

Cell-free DNA analysis

Quality Control of Cell-free DNA Samples
Analyzed with Next-Generation SequencingiApplication note
cfDNA samples

Mono-

500

Average region size
353 bpA. Sample
no. 5

Up

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w
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A. Sample
no. 5

Sample Intensity [Normalized FU]

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B. Sample
no. 8

Lo

35

Size
[bp]

%cfDNA = 83%

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w
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Size
[bp]

50

0

0

B. Sample
no. 8

600
Sample Intensity [Normalized FU]

120

100

80

60

40

Di-

Tri-

Average region size
436 bp

500
400
300
200
100

20

Read the full application note

1500

1000

700

500

400

300

200

1000

700

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500

400

300

200

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100

75

50

35

Size
[bp]

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140

400

25

Sample Intensity [Normalized FU]

4000

Sample Intensity [Normalized FU]

Cell-free DNA (cfDNA) has become an important input material for
NGS as a result of the noninvasive collection methods from bloodand urine. However, analysis of cfDNA can introduce challenges
due to its low concentration and possible contamination from highmolecular weight DNA, both necessitating reliable quality control.
The Agilent 4200 TapeStation system is a vital quality control(QC) tool in NGS workflows. The TapeStation systems and Cellfree DNA ScreenTape assay provide a %cfDNA quality metric fordetermining the quality of input cfDNA for downstream processes.
As part of the German Cancer Research Center (Deutsches
Krebsforschungszentrum), the Sample Processing Lab collaborated
with the Genomics and Proteomics Core Facility to track 13 cfDNA
samples with the TapeStation system, from initial QC with the%cfDNA metric (see Figure), through the NGS workflow, to the final
sequencing results. All 13 samples reported a %cfDNA greater than
83%, with successful library preparation and sequencing metrics.

%cfDNA = 97%

er

Abstract

Final NGS libraries

Size
[bp]

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Use of the Agilent 4200 TapeStation System
for Sample Quality Control in the Whole Exome
Sequencing Workflow at the German Cancer
Research Center (DKFZ)

The red lines indicate the recommend size range (250 to 350 bp).The
analysis of the final NGS libraries with the 4200 TapeStation system
confirmed successful DNA library preparation for all 80 samples andthe six positive control samples.

F5B

G5 H5 A6 B6 C6 D6 E6

1,500
1,000
700
500
400
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200

5,000
Sample intensity [FU]

100

268

Upper

4,000
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D

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C

Lower

25

The 4200 TapeStation system was used for quality control of the
final NGS libraries. These were expected to be sized between 250
and 350 bp with a minimum concentration of 2 ng/μL. In a gel viewof 15 samples, lane B5 and D6 show negative controls (A). B shows
an example of an electropherogram of one sample. The distributionof the concentration for all 80 samples plus eight controls is
illustrated in C. The two positive controls are shown as green
symbols. The red lines indicate the recommended concentrationthreshold (2 ng/μL). The maximum peak size for all 80 samples plus
eight controls is displayed in D. The two positive controls are shownas green symbols.

[bp] L3 (L) G4 H4 A5 B5 C5 D5 E5Size (bp)

Abstract

A

Concentration (ng/µL)

Application note

i

50
0

20

40

Sample

60

80

100

0

0

20

Read the full application note

40

Sample

60

80

100

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Evaluating the Agilent 4200 TapeStation System
for High Throughput Sequencing Quality ControliApplication note
Abstract
Quality control of NGS libraries is key to the success of any
sequencing run. The D1000 ScreenTape and High Sensitivity
D1000 ScreenTape assays can be used for quality control,
providing DNA sizing, and quantification. The bar chart shows
the sizing results of a sample in 3 dilutions analyzed with theHigh Sensitivity D1000 ScreenTape assay on both the 2200 and
4200 TapeStation platforms. DNA concentration determinedwith the 4200 TapeStation system is plotted against theconcentration measured with the 2200 TapeStation system for
the High Sensitivity D1000 ScreenTape assay.

A

The data demonstrates that results obtained with the Agilent
High Sensitivity D1000 assay using both TapeStation systems
are directly comparable and highly reproducible.

B 700

Marker500

Sample intensity (FU)400

Marker300

DNA sample C
200
1:25
1:30

700

1,000
1,500

500

300

200

100

Agilent 2200
TapeStation system

400
300
200
100
1:25

1:30

1:36

DNA sample C

Agilent 2200 TapeStation
concentration [pg/µL]

Agilent 4200
TapeStation system

500

Size [bp]Size [bp]

C

600

0

50

0

25

0

400

1:36

100

DNA sample C
DNA sample D

1,000
900
800

y = 1.0344x
R2= 0.9832

700
600
500
500

600

700

800

900 1,000

Agilent 4200 TapeStation concentration [pg/µL]

Read the full application note

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Quality Control for Agilent SureSelect QXTWGS Library PreparationiApplication note
Abstract
For multiplex sequencing, SureSelect whole-genome libraries
are pooled so that each index-tagged sample is present inequimolar amounts in the final pool. The 4200 TapeStation and2100 Bioanalyzer systems provide molarity and quantification data
along with the sizing information in the region table of the software.
For each library generated by various gDNA input amounts, themolarity was plotted in a graph comparing both systems. The datasummarized in the table demonstrates that sizing and quantificationof amplified libraries with the High Sensitivity D5000 ScreenTape
assay match the results of the High Sensitivity DNA assay of the2100 Bioanalyzer system.
QXT

Agilent 4200 TapeStation
Agilent 2100 Bioanalyzer

2,500

Molarity (pmol/L)

2,000

1,500

1,000

500

0

20 ng Male DNA

50 ng Male DNA

Starting material

80 ng Male DNA

Average size (bp)Region molarity (pmol/L)

Agilent 4200 TapeStation Agilent 2100 Bioanalyzer Agilent 4200 TapeStation Agilent 2100 Bioanalyzer
SystemSystemSystemSystem
20 ng
50 ng
80 ng

mean

519

533

850

% CV

1.2

2.8

5.4

0.6

mean

849

868

1513

1473

% CV

0.6

1.4

1.7

4.5

mean

1065

1157

2073

2097

% CV

3.8

1.1

4.3

5.5

Read the full application note817

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Sample Quality Control inAgilent NGS SolutionsiApplication note

500

HaloPlex

400
300
200

1,000
1,500

700

500

300

0

100

100
Size
(bp)

Sample intensity (normalized FU)

Lo

Up

w

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25

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200

HaloPlex

150
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35 100 200 300 400 600 1,000 2,000 10,380
Size (bp)

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HaloPlex HS

2,000
1,500
1,000

1,000
1,500

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500

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0

25

500
Size
(bp)

Sample intensity (normalized FU)

Sample intensity (normalized FU)

HaloPlex and HaloPlex HS target enrichment technology uses
an amplicon-based approach. The final libraries of HaloPlex and
HaloPlex HS workflows show a profile with a characteristic smearin the range of 175 to 625 bp, (A) and (B), and 190 to 545 bp
respectively (C) and (D). The appearance of the profile may vary
due to specific library designs and the overall quality of the input
material. The electropherogram should be checked for the presenceof artefactual peaks with sizes less than 150 bp, as these are related
to primer dimers that can cluster and consume sequencing capacity.
If the primer dimer peak is greater than 10% of the total product, an
additional cleanup step with AMPure beads is recommended.

A

Sample intensity (normalized FU)

Abstract

200
150

HaloPlex HS

100
50
0
35

150

Read the full application note

300

500 1,000
Size (bp)10,380Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Quality Control in Illumina Sequencing
Workflows Using the TapeStation System

4
3
2
1

pe
r
Up

4
3
2
1

1,000
1,500

700

300
400
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100

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25

0

Read the full application note

Size
(bp)

700

25

28
6

w
5

1,000
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(bp)

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50

NGS target enrichment enables a detailed analysis of specificregions to identify causal genetic variants of complex conditions.
The SureSelect XT protocol is designed to create libraries withenriched targeted regions of the genome for sequencing with Illumina
platforms. The two intermediate QC steps include evaluation of asmear size after shearing (A) and before capturing (B). These steps
can be carried out using the D1000 ScreenTape assay. The expected
size range of the maximum peak of sheared DNA is 150 to 200 bp.
For precapture library, a larger maximum peak size of 225 to 275 bp
is expected due to adapter ligation. The last QC step qualifies the final
library before pooling (C). Another size shift is expected as a resultof adding index sequences. The peak maximum of the final library is
expected to be between 250 and 350 bp. A minimum concentration of
2 ng/μL is expected for successfully generated final libraries.

×103

25

Abstract

A
Sample intensity (normalized FU)

Application note

i

Size
(bp)

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

250

300 400

600

0

100

C
B. HS Small
Fragment kit, ultrashort array, sample #1

6,000

3,000

1

UM

Size (bp)F
D. HS NGS
Fragment kit, ultrashort array, sample #3

HS Small kit ultrashort array Sample #1

2,800 HS NGS kit ultrashort array Sample #3
LM
2,400

UM

LM

326

1,500

Size (bp)

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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
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RFU

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1

9

300 400

HS NGS kit short array Sample #3
UM

153

800

8

200

C. HS NGS
Fragment kit, short array, sample #3
E

1,000

RFU

2,000
1,600
325

1,200
1,0.8

153

UM

0.4

Read the full application note

6,000

3,000

500

400

Size (bp)

600
700
800
1,000
1,500

Size (bp)

300

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HS Small kit short array Sample #1
LM

900 1,200
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600
UM
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8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Time (min)

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6,000

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LM

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RFU

The quality of NGS libraries is crucial to successful sequencing
results. The Fragment Analyzer systems offer easy analysis ofsheared genomic DNA (gDNA) and libraries with the HS NGS
Fragment kit (1-6000 bp) and the HS Small Fragment kit. The HS
NGS Fragment kit (C and D) analyzes larger smears and fragments
up to 6,000 bp, while the HS Small Fragment kit (A and B) focuseson smaller sizes up to 1,500 bp. The FA 12-Capillary Array Ultrashort
22 cm decreases run time by 10 to 20 minutes compared tothe standard FA 12-Capillary Array Short, 33 cm. The size andconcentration of several DNA smears were compared between
both kits and the short and ultrashort arrays. Library sizing andquantification remained consistent between the short (A and C) andultrashort (B and D) arrays and the two kits. The HS Small Fragment
kit and the HS NGS Fragment kit can be used interchangeably forsizing and quantification of NGS libraries, as long as the sample fits
within the sizing range of the kit.

HS NGS kit ladder ultrashort array

A. HS Small
Fragment kit, short array, sample #1
B

RFU

AbstractD

HS Small kit ladder ultrashort array

1

Application note

4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0

A

RFU

RFU

Comparison of the Agilent HS Small Fragment Kit
and Agilent HS NGS Fragment Kit on the
Fragment Analyzer Systems

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Monitoring Library Preparation for Next-Generation
Sequencing in Systems Biology Omics Analysisi

Application note
1,
00
Up 0
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700

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Size (bp)

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100

0

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300

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10

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40

12

100

60

14

50

80

B

25

Sample intensity (normalized FU)

100

15
50

Frequently, the first step of a library preparation protocol is thefragmentation of gDNA by shearing with an ultrasonicator. Optimal
shearing in NGS workflows can be verified by evaluating the size
distribution and electropherogram pattern of fragmented DNA
samples using the 2100 Bioanalyzer (A) and the 4150 TapeStation
systems (B) with the DNA 1000 kit and D1000 ScreenTape assays,
respectively. Electropherograms of sheared DNA in this example
display an even size distribution with no undesirable shouldering.
The fragmented DNA samples show a maximum peak size between
260 and 310 bp on both systems, verifying optimal shearing (C).
The size of the sample at this QC step can be compared to the sizeof sample after adapter ligation in the library preparation workflow,
at which point a shift in size is expected. Overall sizing results ofthe 2100 Bioanalyzer and the 4150 TapeStation systems correlated
highly with an average deviation of 2.2% for all 30 samples analyzed.

×103
16

120

Size (bp)C

400

DNA 1000 assay
D1000 ScreenTape assay

350
300
Average size (bp)

Abstract

A

10
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1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Sample number

Read the full application note

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis
[FU]
[FU]

Quality Control of NGS Libraries
with Daisy Chains

A

A

300

i

300
250
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200
200
150
150

Application note

100
100
50
50
0

8000

Sample Intensity [Normalized FU]

250
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400

B

7000
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1400

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2200
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250

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50

Read the full application note

10000

5000

3500

2500

1500

1000

400

600

600

500

400

300

Size
[bp]

250

1

20

20

2800
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Size
[bp]

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3500

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15
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1

C

200

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[bp]

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Size
[bp]

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6000

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C

300

7000

[bp]

400

3400

10380

RFU

B

2000

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500

250

400

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300

15

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1500

Sample Intensity [Normalized FU]

8000

100

100

0
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[bp]

[bp]

1500

A

100

Sample Intensity [Normalized FU]

35
35

300

RFU

The Bioanalyzer, TapeStation, and Fragment Analyzer systems withdedicated high sensitivity assays allow unambiguous detectionof daisy chains and accurate sizing of the target library peak. Two
KAPA HyperPlus libraries with different levels of amplification wereanalyzed on the Bioanalyzer (A), TapeStation (B), and Fragment
Analyzer (C) systems using the High Sensitivity DNA kit, HighSensitivity D5000 ScreenTape assay, and HS Small Fragment kit
respectively. As shown in the overlays, both libraries (blue – library 1,
red – library 2) contained the desired library peak with a pronounced
secondary peak in library 2. The excessive amplification of library 2
resulted in the formation of daisy chains, which were observed as
an additional higher molecular weight peak in all electropherograms.
The daisy chains migrated slower through the gel matrix andwere easily detected by all Agilent automated electrophoresis
instruments using the respective assays. In this application note,
we provide a recommendation for which assay to employ to reliably
visualize daisy chain products in next-generation sequencing
libraries. Furthermore, we emphasize the consistency between the
instruments and reproducibility of analysis confirmed by results on aseries of double dilutions.

[FU]

700
800
900
1000

Abstract

0

Size
[bp]

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Quality Control of Magnis SureSelect XT HS
Workflows with Agilent AutomatedElectrophoresis Solutions

Average Size Post-Shear Library
200
150

Application note

100
50

Abstract

0A

Size in bp

NA18507

1000

800

B

Average Size Post-Shear
LibraryHD803
HD799

NA 18507 (10X)

Average Size Pre-Capture Library

200
400
150
300
100
200
50
100
00

600

NA 18507 (10X)

HD798 HD799 HD799

HD803
HD803

Size in bp

AverageSize
SizeofPre-Capture
Library
Average
Post-Capture
Library

400

200

HD803

7000

15000
48500

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250

0

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200
100
100
0
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HD798

HD799
100

Sample Intensity (Normalized FU)

The Magnis NGS Prep system is an automated NGS library
preparation solution for the SureSelect XT HS system. It addresses
challenges of manual library preparation, such as hands-on time,
expertise, optimization, and validation for diverse applications.
Performing QC steps and quantification on the starting material, thematerials derived from intermediate steps (optional), and the final
library is beneficial in ensuring reliability and overall success of thesequencing data. QC steps can be performed with the automatedelectrophoresis portfolio of instruments, including the Bioanalyzer,
TapeStation, and Fragment Analyzer systems. gDNA was assessedwith the Genomic DNA ScreenTape assay on the 4150 TapeStation
system for overall integrity and size in high-quality gDNA, and mildly,
moderately, and highly degraded FFPE samples (A). This assay
applies a quality score, the DNA integrity number (DIN), to eachsample. The score is used to optimize the fragmentation step anddetermine the amount of input DNA to be used in library preparation.
Post-capture libraries were assessed on all three platforms anddisplayed similar sizing (B).

i

Size
[bp]

NA 18507
18507
NA

HD798
HD798

HD799
HD799

HD803
HD803

4150 TapeStation
2100ofBioanalyzer
5200 Fragment
Average Size
Post-Capture
LibraryAnalyzer
400
300
200

Read the full application note
1000

NA 18507
4150 TapeStation

HD798
2100 Bioanalyzer

HD799

HD803

5200 Fragment Analyzer

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Comparison of DNA Sample QC for NGS
Workflows with the Agilent Fragment
Analyzer and Bioanalyzer SystemsiTechnical overview
10447

Abstract

10000

In this technical overview, precapture and final libraries werecompared on both the Agilent Fragment Analyzer and Bioanalyzer
systems with their corresponding DNA assays to demonstrate
data equivalency across platforms. The DNA assays selected have
similar analytical specifications, allowing for seamless comparison
between the instruments. Both systems support standard-sensitivity(SS) and high-sensitivity (HS) kits, covering a wide range of sample
concentrations that occur at different QC checkpoints throughout
library preparation. Starting DNA material can vary greatly inconcentration, depending on whether its source is from freshmaterial or ancient samples. The HS kits are ideal for conservation of
low-concentration samples, and can be used at all recommended QC
checkpoints. Alternatively, the SS kit has the advantage of analyzing
high-concentration samples while eliminating time-consuming
dilution steps. Having the option of both SS and HS kits simplifies
the library preparation workflow. The SS and HS kits are compared inthis technical overview.

8500

A. Fragment
AnalyzerAnalyzer
A.
Fragment
NGS final library no. 22
NGS final library no. 22

9500

UM

9000

8000
LM

7500

420 bp

6500
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Adapter dimer

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2423

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1

RFU

7000

Size (bp)[FU]

B.
Bioanalyzer
B. Bioanalyzer
NGS final library no. 22
NGS final library no. 22

UM

250

200
LM

413 bp150

100

Adapter dimer
500Region 1
35

100

150

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300

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500

500

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Read the full technical overview

10380

[bp]

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Preventive Control of Sequencing
Through the Insert with the Agilent
5200 Fragment Analyzer Systemi
B

Application note

2698
2400
2200

Range

ng/µL

% Totalnmole/L

Avg. Size%CV

A2: S1 - 2 ng/µL

110 bp to 1350 bp

2.3296

98.7

11.5454

332

23.30

1600

220 bp to 1350 bp

2.233994.6

10.8748

338

21.67

1400

286 bp to 1350 bp

1.741273.7

7.9547

360

18.82

RFU

1800

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2698

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100

2400

1

Size (bp)

1400

C

1200
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LM

2000

400

1800

200
RFU

6000

3000

700
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900
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Size (bp)

Read the full application note

6000

3000

700
800
900
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200

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300

A

1

The Agilent Fragment Analyzer systems, together with the Agilent
NGS Fragment kits and the companion Agilent ProSize data analysis
software, offer a well-established solution for sample quality control(QC) in next-generation sequencing (NGS) workflows. This applicationnote expands the capabilities of the ProSize smear analysis, anddemonstrates how its functionality can be further applied to avert
a specific sequencing issue called sequencing through the insert.
This data is then introduced into the sequencing read and, in somecases, can increase the background noise, decreasing the qualityof the overall sequencing run. The percent total value of the ProSize
software allows a user to estimate the percentage of the librarythat will be sequenced through the insert based on the planned
sequencing run method and electrophoretic profile of the library. Easy
and comprehensive smear analysis enables the user to determine an
optimal read length, and thereby minimize the number of unwantedbases present in the sequencing reads, saving expensive reagentsand time for auxiliary data processing.

2000

ID

RFU

Abstract

UM

LM

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Quality Assessment of NGS Libraries using
Agilent Automated Electrophoresis SystemsiTechnical overview
Abstract
A.

A.

Sizing Comparison
500
450
400
350
300
250
200
150
100
50
0

Quantification Comparison
700

Concentration (pg/µl)

Size (bp)

The Agilent automated electrophoresis instruments, including
the Bioanalyzer, Fragment Analyzer, and TapeStation systems,
are well-suited for QC of NGS libraries, providing high-quality
quantification, qualification, and sizing of nucleic acids toallow for confident assessment of samples throughout the
NGS workflow. Each instrument offers specific benefits suited
to meet a variety of individual laboratory needs, such as
throughput, sensitivity, speed, and resolution. To demonstrate
the equivalency of the systems to each other, NGS libraries
were compared across each instrument. Each instrument
offers a versatile assay portfolio that covers broad sizing andconcentration ranges. The DNA analysis kits chosen for analysisof the NGS libraries in this technical overview have similar
specifications, with a sizing range well-suited for the NGS smear,
allowing for comparison between the instruments. This technicaloverview highlights the capability of each system to accurately
size and quantify NGS libraries.

Library 1

600
500
400
300
200
100
0

Library 2

Library 1

Library 2

Bioanalyzer High Sensitivity DNA

Fragment Analyzer High Sensitivity NGS

Bioanalyzer High Sensitivity DNA

Fragment Analyzer High Sensitivity NGS

Fragment Analyzer High Sensitivity Small Fragment

TapeStation High Sensitivity D1000

Fragment Analyzer High Sensitivity Small Fragment

TapeStation High Sensitivity D1000

B.

B.
Average Size (bp)Instrument and Kit%CV

Library 1

Library 2

Library 1

Library 2

Bioanalyzer High Sensitivity DNA

431

470

0.35

0.43

Fragment Analyzer High Sensitivity NGS

419

448

0.96

0.22

Fragment Analyzer High Sensitivity Small Fragment

429

457

0.54

0.25

TapeStation High Sensitivity D1000

423

446

1.54

0.22

Average Conc.
(pg/µL)
Instrument and Kit
Bioanalyzer High Sensitivity DNA%CV%error

Library
1

Library
2

Library
1

Library
2

Library
1

Library
2

469

464

3.95

2.45

17.20

17.74

Fragment Analyzer High Sensitivity NGS

487

479

10.86

14.41

13.99

15.13

Fragment Analyzer High Sensitivity Small Fragment

482

491

14.56

7.81

14.84

12.91

TapeStation High Sensitivity D1000

533

549

7.91

7.05

5.77

2.60

Read the full technical overview

Introduction

QC Instruments

Sample Types

NGS Workflow Solutions

NGS library analysis

Detection of Adapter Dimers in NGS Libraries
with the Agilent Fragment Analyzer and
TapeStation SystemsiTechnical overview
Abstract
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. Even small amountsof adapter dimers in a library can contribute to detrimental sequencing
results. The presence of adapter dimers can affect the accuracy oflibrary quantification, leading to suboptimal flow cell loading and reduced
clustering efficiency. Smaller fragments, such as dimers, are alsopreferentially sequenced over larger fragments, so even low levels ofadapter dimers could contribute to issues such as reduced output, lower
diversity, and decreased genome coverage. While the optimal amount ofadapter dimer in a library is zero, there are thresholds of adapter dimer ina library that may be present and still generate satisfactory sequencing
results. Thus, it is highly recommended to minimize and remove adapter
dimers from the library before sequencing. This technical overview
highlights the sensitivity and resolution of the Agilent Fragment Analyzer
and TapeStation systems to detect adapter dimers even lower than therecommended threshold of 0.5% and deliver an accurate assessment of
the percentage of adapter dimer in the library.

0.5% Fragment

0.5% Fragment

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.

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

Read the full technical overview

Introduction

QC Instruments

Want to Explore Even More Resources?
Performing quality control of your nucleic acid samples with Agilent automatedelectrophoresis solutions can help advance your next-generation sequencing workflows
while conserving valuable time and resources.
Browse all of our automated electrophoresis solutions at:www.agilent.com/en/product/automated-electrophoresis
For additional information on NGS sample quality control solutions from Agilent, visit us at:www.agilent.com/en/solutions/genomics-applications-solutions/sample-QC/ngs
Buy online:
www.agilent.com/chem/store

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For Research Use Only. Not for use in diagnostic procedures.
PR7001-1876© Agilent Technologies, Inc. 2024
Published in the USA, January 1, 2024
5994-6990EN

Sample Types

NGS Workflow Solutions