Application NoteGenomics

Quality Control of Cell-free DNA
Samples Analyzed with
Next-Generation Sequencing
Authors

Abstract

Christina Geörg and Katrin Pfütze,Sample Processing Lab,
German Cancer Research Center DKFZ,Heidelberg, Germany

Next-generation sequencing (NGS) is an essential tool for analysis of biosamples
in clinical research. Cell-free DNA (cfDNA) has become an important input materialfor NGS as a result of the noninvasive collection methods from blood and urine.
However, analysis of cfDNA can introduce challenges due to its low concentrationand possible contamination from high-molecular weight (HMW) DNA, bothnecessitating reliable quality control. The Agilent 4200 TapeStation system is a vitalquality control (QC) tool in NGS workflows. The TapeStation systems and Cell-free
DNA ScreenTape assay provide a %cfDNA quality metric for determining the qualityof input cfDNA for downstream processes. The Sample Processing Lab (SPL)
collaborated with the Genomics and Proteomics Core Facility (GPCF), both part ofthe German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ)
to track 13 cfDNA samples with the TapeStation system from initial QC with the%cfDNA metric, through the NGS workflow, to the final sequencing results. All 13
samples reported a %cfDNA greater than 83% with successful library preparationand sequencing metrics.

Ute Ernst, Nicolle Diessl, andMarie Beckhaus
Genomics and Proteomics Core Facility,
German Cancer Research Center DKFZ,Heidelberg, Germany
Elisa Viering and Chava Pocernich,Agilent Technologies, Inc.

Introduction

Liquid biopsy is a noninvasive way to collect samples frombiofluids such as plasma, serum, urine, and cerebrospinal
fluid for serial assessments. The cfDNA found in thesebiofluids is often DNA released from apoptotic cells. The DNA
is fragmented between the histone complexes into various
sized cfDNA. The different cfDNA fragments are referred toas the mono-nucleosome, di-nucleosome, and tri-nucleosome
peaks. The majority of cfDNA is in the mono-nucleosome
fragment, around 170 bp in length. cfDNA is often used forscreening, following disease progression, and monitoring
effectiveness of disease treatment. Sequencing cfDNA canprovide information on tumor-derived mutations since asmall fraction of the cfDNA is circulating tumor DNA (ctDNA),
derived from tumor cells. The genetic information from ctDNA
contains biomarkers that can be used to monitor cancer
progression.
The SPL provides expertise in the isolation of DNA, RNA, andproteins and quality control analysis of these extractions fromdifferent sources of material such as tissue biopsies, FFPE
tissue blocks, blood, and cells. The SPL works closely with theGPCF, transferring suitable samples for sequencing. Currently,
the SPL is developing and optimizing extraction procedures ofcirculating cell-free tumor DNA from liquid biopsies, including
automated extraction processes.
It is essential to know the quality of incoming samples for theexperimental success of library preparation and sequencing.
HMW DNA in the cfDNA sample may interfere with libraryyield and sequencing quality. After extraction of cfDNA, theSPL performed cfDNA sample quality control with the 4200
TapeStation system and the Cell-free DNA ScreenTape. The
Cell-free DNA ScreenTape assay was developed specificallyfor cfDNA analysis. They investigated the %cfDNA quality
metric¹, which was introduced with the Cell-free DNA
ScreenTape assay as a way to screen the quality of cfDNA
samples for NGS library preparation and sequencing.

2Experimental

The SPL received blood collected in Fusion2 cfDNA BCT(Hiss Diagnostics p/n 230256) and PAXgene Blood ccfDNA(PreAnalytiX, a Qiagen/BD company, p/n 768115) collection
tubes. cfDNA was extracted from 8 to 9 mL of plasma withthe QIAmp MinElute ccfDNA Midi kit (Qiagen, p/n 55284)
and eluted in 50 µL of water. Quality control analysis wasperformed with the Agilent 4200 TapeStation system andthe Agilent Cell-free DNA ScreenTape (p/n 5067-5630) andCell-free DNA reagents (p/n 5067-5631). Concentration wasdetermined with both the Qubit and the dsDNA HS assay(p/n Q32851) and the 4200 TapeStation and Cell-free DNA
assay. Libraries were prepared with the Agilent SureSelectXT
Low Input Target Enrichment system (p/n 5191-4080) with
25 or 50 ng of input cfDNA. Final libraries went through QC
analysis with the 4200 TapeStation and the HS D1000 assay(ScreenTapes p/n 5067-5584, reagents p/n 5067-5585).
Sequencing was done with the Illumina HiSeq 4000 system
and a paired-end 100 bp run. Sequencing data QC thresholds
are mostly based on Illumina specifications.

Mono-Sample Intensity [Normalized FU]

4000

3000

2000
Di1000

1000

700

1000

600

500

400

300

200

150

100

7550Size[bp]07050wLo140

Size[bp]%cfDNA = 83%er

350Quantification

B. Sample
no. 8

Mono-Sample Intensity [Normalized FU]

120

100

8060

40Di-Tri-

20

700

600

500

400

300

200

150

10075

50

350Figure 1. cfDNA samples analyzed with the Agilent 4200 TapeStation system andthe Cell-free DNA ScreenTape assay. (A) Sample no. 5 displayed the mono- and dinucleosome peak. (B) Sample no. 8 displayed the mono-, di-, and tri-nucleosomepeaks.

cfDNA Mono-nucleosome Size
210
190
170150Size (bp)

Total cfDNA concentration was measured on the 4200
TapeStation system with the Cell-free DNA ScreenTape
assay and on the Qubit with the dsDNA HS assay. Qubit
has two assays available for measuring low-concentration
DNA samples: the dsDNA HS assay (p/n Q32851) and thenewer 1X dsDNA HS assay (p/n Q33230). Previously, weshowed that total cfDNA concentrations measured withthe Cell-free DNA ScreenTape assay are highly comparablewith the Qubit 1X dsDNA HS assay, ddPCR, and qPCR, while
the Qubit dsDNA HS assay consistently reported higher
concentrations².

0

A. Sample
no. 5

SizingThirteen cfDNA samples were screened for QC on the 4200
TapeStation system with the Cell-free DNA ScreenTape
assay, which was developed specifically for cfDNA analysis.
The electrophoresis separations with the Cell-free DNA
ScreenTape assay all displayed the mono- and di-nucleosomepeaks (Figure 1A) with the majority of samples showing thetri-nucleosome peak (Figure 1B). It is common with cfDNAsamples to observe sample to sample variation regarding thenumber of nucleosome fragments present. The TapeStation
analysis software automatically reported a size for themono-nucleosome peak. Sizing of the mono-nucleosome
peak across all the samples was similar, ranging from 170 to195 bp (Figure 2).

70w
erLo

Quality control of extracted cfDNA%cfDNA = 97%

50

Results and discussion

130
110
90
7050
12345678910111213Sample NumberFigure 2. Mono-nucleosome fragment size from cfDNA samples analyzed with
the Agilent 4200 TapeStation system and the Cell-free DNA ScreenTape assay.

3

The TapeStation system has the distinct advantage ofseparation analysis, allowing for sizing and quantificationof the different components that make up a sample. Region
analysis set by the user provides detailed information about
each component of interest in the total sample. Concentrationof the cfDNA portion of the sample was reported by region
analysis, set at 50 to 700 bp, and compared to the total
sample (Figure 4). There is no way for the Qubit to discern
the concentration of the different components of a sample
since it does not perform any type of separation and can only
measure the total concentration of a sample.

cfDNA Concentration Comparison
4035
Concentration (ng/µL)

In this current study, quantification of the total cfDNA
samples was consistently higher with the Qubit dsDNA HS
assay compared to the Cell-free DNA ScreenTape assay(Figure 3), consistent with the previous studies.

3025201510501235478910111213

TapeStationQubitFigure 3. Total cfDNA sample concentration was measured on the Agilent 4200
TapeStation system with the Cell-free DNA Screen Tape assay and on the Qubit
with the dsDNA HS assay.

Total Sample vs cfDNA Concentration
30Concentration (ng/µL)

25
20
15
10
50123

4

5

67

8

910

11

1213Sample Number
Total sample

cfDNAFigure 4. The Agilent 4200 TapeStation system with the Cell-free DNA Screen
Tape assay measured both the total sample concentration and the cfDNA of thesample by region analysis set at 50 to 700 bp, allowing for comparison to the total
sample concentration.

Library preparation%cfDNA
10095%cfDNA

All samples continued through library preparation. Due tothe low concentration available from cfDNA extraction,
samples no. 3, 8, and 9 had a lower input of approximately 25ng cfDNA for library preparation compared to 50 ng for themajority of samples. A threshold of 10 ng sample input had
previously been established for the SureSelectXT Low Input
Target Enrichment system³ by the DKFZ. The number of PCR
cycles was adapted to the input amount and fragmentation
was excluded due to the small fragment size of cfDNA.
DKFZ did not incorporate size selection to eliminate HMW
DNA, since the %cfDNA was high for all samples, indicating
minimal HMW DNA present.

6Sample Number

Percent cfDNA
The %cfDNA metric was developed for the TapeStation
instrument as an easy-to-use screening tool for determining
the quality of cfDNA samples for NGS library preparation andsequencing. The %cfDNA metric reports the percent of cfDNA
fragments from 50 to 700 bp in the total sample. This range
encompasses the mono-, di-, and tri-nucleosome cfDNAfragments. The metric ranges from 0 to 100%. HMW DNA
can be extracted with cfDNA and interfere with downstream
processes. A high percent cfDNA indicates a high-quality
sample with very little HMW DNA. Likewise, a low %cfDNA
indicates the presence of HMW DNA. All the samples fromthe SPL reported a %cfDNA between 83 to 97% cfDNA,indicating minimal HMW DNA present (Figure 5).

49085807512345678910111213Sample NumberFigure 5. %cfDNA metric for samples analyzed with the Agilent 4200 TapeStation
system and the Cell-free DNA Screen Tape assay. All samples reported a %cfDNA
above 83%.

Upperw
er
LoSample Intensity [Normalized FU]

400

300

200

100

1500

Size[bp]w
er

1000

700

500

400

300

200

100

5025Lower
Lo

700

B. Sample
no. 8600

Average region size
436 bp

500
400
300
200
100Size[bp]

1500

1000

700

500

400

300

200

50

100025The library concentration was compared on the Qubit andTapeStation. All but one sample measured below the 2 ng/µLlibrary concentration threshold on the TapeStation. Sampleno. 11 reported exactly at 2 ng/µL. The varied amount of input
cfDNA did not affect the final library concentration, as alllibraries reported a similar concentration.

Average region size
353 bpA. Sample
no. 50Sample Intensity [Normalized FU]

The library size was assessed by region analysis, taking into
consideration the entire library sample. All samples except no.
8 fell within the library size QC threshold of 200 to
400 bp (Figure 7). Sample no. 8 was slightly outside therange, at 436 bp. It also reported the lowest %cfDNA, at 83%,indicating the presence of a small amount of HMW DNA.
Samples with slightly higher %cfDNA at 85% fell within thelibrary size QC threshold. All samples were below the required
0.5% adapter-dimer (data not shown).

500

Figure 6. Library samples analyzed with the Agilent 4200 TapeStation system andthe HS D1000 assay. (A) sample no. 5 (B) sample no. 8. The three peaks represent
the mono-, di-, and tri-nucleosome cfDNA peaks with adapters attached.

Average Library Region Size
500450400350
Size (bp)

QC was performed again after library preparation with theTapeStation and the HS D1000 assay to ensure quality
libraries. All libraries displayed a shape similar to the cfDNAtrace, with most showing three nucleosome peaks or asmear in the region of the tri-nucleosome peak (Figure 6).
For example, cfDNA sample no. 5 (Figure 1A) displayed twopeaks and only a small smear in the tri-nucleosome region,
which was mimicked in the library trace. Library QC required
the smear size to be between 200 to 400 bp, a concentrationof 2 ng/µL, and with less than 0.5% adapter-dimer present for
patterned flow cell.

30025020015010050012345678910111213Sample NumberFigure 7. Average region size of the libraries was assessed with the Agilent 4200
TapeStation system and the HS D1000 assay with the region analysis setting.

5Library Concentration
3.53Concentration (ng/µL)

In addition, all samples reported comparable concentrations
between the Qubit and TapeStation, except sample no. 3,
which reported a much higher concentration on the Qubit
dsDNA HS assay (Figure 8). Excluding sample no. 3, thepercent difference from the Qubit ranged from 0.35 to 15%,
averaging 6.7% difference, demonstrating comparable results
between the two instruments. The percent difference waswell within the range of the HS D1000 assay and all samples
continued through to sequencing.

2.5
21.5
10.5
01

2

3

45

Sequencing QC metrics
Table 1 summarizes and compares the sequencing QC
metrics of all 13 samples. Sequencing QC thresholds for
cfDNA have not yet been defined due to the lack of statisticalinformation, but the threshold for OnTarget rate > 50% has
been set for the SureSelect XT HS Human ALL Exon v7
protocol. General thresholds for successful sequencing,including mapping rate > 99%, duplicates </= 20%, and insert
size > 140 bp, were followed. Only three samples reported
one of the sequencing metrics slightly outside the thresholds
listed. The mapping rate for all 13 samples was 99.94%
or higher. Only sample no. 3 had an OnTarget rate below
50%, reporting at 44%. Samples no. 8 and 9 reported a 21%
duplicate rate slightly outside the threshold, possibly due
to PCR or patterned flow cell. Successful sequencing wasobtained for all 13 samples based on the reported sequencing
metrics. These results demonstrated that a sample withgreater than 83% cfDNA can lead to successful library
preparation and sequencing. Samples with a %cfDNA lower
than 83% were not a part of this study and have not been
evaluated to determine if they are viable samples for library
preparation and meaningful sequencing results. Additional
samples with a wide range of %cfDNA need to be evaluatedin order to provide a more robust understanding of how the%cfDNA metric relates to sequencing success. The DKFZ iscontinuing to fine tune protocols in order to set specifications
for library preparation of cfDNA samples that would lead tosuccessful sequencing.

6678910111213Sample Number
TapeStation

Qubit

Figure 8. Library concentration was analyzed with the Agilent 4200 TapeStation
system and the HS D1000 assay. The 4200 TapeStation system and Qubit
concentration were very similar for all but sample no. 3.

Table 1. Sequencing quality control metrics and %cfDNA reported for all
13 samples.
Sequencing QC Metrics
SampleNumberMapped[%]Duplicates[%]

On Target[%]R2%>=Q30[%]

Insertsize [bp]% cfDNA

1

99.97

16.4

67.2

91.4

166

92

2

99.98

13.3

69.4

91.4

166

90

3

99.96

15.4

44.7

88.8

166

85

4

99.97

15.0

60.5

90.5

166

86

5

99.97

15.0

60.5

90.5

166

97

6

99.96

12.0

68.2

91.4

166

89

7

99.96

11.8

68.8

91.4

167

91

8

99.94

21.3

55.7

88.8

167

83

9

99.95

21.2

51.4

88.8

166

91

10

99.96

11.8

64.591.4

166

89

1199.97

9.8

68.7

91.4

167

89

1299.97

11.7

67.8

91.4

166

98

Conclusion

The TapeStation systems offer a Cell-free DNA ScreenTape
specifically designed for cfDNA samples. This assay
overcomes the complexity of cfDNA sample. The sensitivityof the assay allows detection of picogram levels of cfDNA
with visualization of the multiple cfDNA monomers. Inaddition, the Cell-free DNA ScreenTape assay quantifies thecfDNA subcomponent of the sample separately from the total
sample. Distinct to the Cell-free DNA ScreenTape assay is the%cfDNA metric, which aids users in determining the amountof HMW DNA in their samples and provides a screening
tool for library preparation. The SPL tracked cfDNA samples
from the initial QC with the %cfDNA metric, through library
preparation, to the end sequencing results. All samples had
a %cfDNA over 83%, with library preparation passing the size
threshold of 200 to 400 bp, but reporting a concentration
below 2 ng/µl, and resulting in successful sequencing.

References
1. Performance Characteristics of the Cell-free DNA
ScreenTape Assay, Agilent Technologies TechnicalOverview, publication number 5994-1390EN, 2019.
2. Cell-free DNA Quality and Quantity Assessment – A
Method Comparison, Agilent Technologies Poster,
publication number PR7000-7364, 2019.
3. SureSelect XT Low Input Target Enrichment System forIllumina Paired-End Multiplexed Sequencing Library forIllumina Multiplexed Sequencing Platforms Protocol,
Agilent Technologies, publication number G9703-90000,
2018.

7

www.agilent.com/genomics/tapestation
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
PR7000-7442© Agilent Technologies, Inc. 2020
Printed in the USA, September, 2020
5994-2284EN