Application Note
Genomics

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

Authors
Christine Voigt
Alacris Theranostics
Eva Graf
Agilent Technologies

Abstract
As part of the CanPathPro Project, Alacris Theranostics (www.alacris.de)investigates the genome and transcriptome of genetically engineered mousemodels. The applied next-generation sequencing protocols are Agilent SureSelectXT
and Illumina TruSeq Stranded Total RNA. In the past, the Agilent 2100 Bioanalyzer
system was used to perform sample quality control for extracted RNA as well as for
DNA and final library quality control (QC) during next-generation sequencing (NGS)
workflows. Quantification of extracted DNA was carried out using a fluorometricquantification method. The Agilent 4150 TapeStation system was evaluated as a
new platform to monitor sample quality during library preparation. The QC stepsof 30 samples were performed in parallel with the 2100 Bioanalyzer system forcomparison.

Introduction
CanPathPro (www.canpathpro.eu), an
EU Horizon 2020 project, is addressing
the challenge of predictive modelingof biological data. During the project,bio-informatic and experimental tools
are being developed and refinedfor the evaluation and control ofmodeling predictions for systems
biology. Within CanPathPro, geneticallyengineered mouse models are used
to follow the temporal molecular andsignaling changes occurring duringcancer development. This includes thehistology of the tumor, proteomics, aswell as the genome and transcriptomeusing next-generation sequencing(NGS). CanPathPro aims to develop
a new systems biology platform forgenerating and testing cancer signaling
hypotheses in biomedical research.
Alacris Theranostics is working in thefield of precision oncology, biomarker
applications, systems biology, and drug
response modeling and co-ordinates the
CanPathPro project.

Experimental
Materials
The HiSeq 2500 system and kits
for sequencing were obtained from
Illumina (San Diego, CA, USA). The
Agilent 4150 TapeStation system(p/n G2992AA) with Agilent Genomic
DNA ScreenTape (p/n 5067-5365) andreagents (p/n 5067‑5366), Agilent D1000
ScreenTape (p/n 5067‑5582) withreagents (p/n 5067‑5583), Agilent
High Sensitivity D1000 ScreenTape(p/n 5067‑5584) with reagents(p/n 5067‑5585), and Agilent RNA
ScreenTape (p/n 5067‑5576) withreagents (p/n 5067-5577) from
Agilent Technologies were used forsample quality control. The Agilent
2100 Bioanalyzer system was usedwith the Agilent RNA 6000 Nano kit(p/n 5067-1511), the Agilent DNA 10002kit (p/n 5067-1504) and the Agilent
High Sensitivity DNA kit (5067-4626).
The Agilent SureSelectXT reagent kit(p/n G9611A) and Agilent SureSelectXT
Mouse All Exon V2 (p/n G9496A) wereused for DNA library preparation. The
Illumina TruSeq Stranded Total RNA
Library Prep Gold (order no. 20020599)
and TruSeq RNA Single Indexes Set A(order no. 20020492) were used for
RNA library preparation. The Qubit 2.0
Fluorometer (Thermofisher Scientific)
with the Qubit dsDNA HS assay kit(order no. Q32854, Fisher Scientific)
and Qubit RNA HS assay kit (order no.
Q32855, Fisher Scientific) was used fornucleic acid quantification. The Covaris
Ultrasonicator S220 (KBiosciences) wasused for shearing. The Thermal Cycler
DNA EngineTetrad 2, BioRad was used
for PCR amplification. Nucleic acids
were extracted with the Qiagen AllPrep
DNA/RNA/miRNA Universal Kit (catalog
number 80224, Qiagen). Unless stated,
all analyses were performed according to
manufacturers’ instructions.

Methods
Sample sources
Samples 1 to 15 were extracted fromtransgenic cancer mouse models fornon-small cell lung cancer (NSCLC) andsamples 16 to 30 from invasive lobular
carcinoma (ILC) of the breast. Eachcancer type is represented by two mousemodels with different mutations inselected cancer-associated genes.

Quality control of starting material
The 4150 TapeStation system with
Genomic DNA ScreenTape assay wasused for sample integrity assessment
and quantification of genomic
DNA (gDNA) starting material. The
quantification of gDNA samples was also
performed using the Qubit system basedon fluorescence detection. Samples werediluted 1:10 before quantification with
the Qubit dsDNA HS assay. Based onthese concentrations, the DNA samples
were normalized to the quantitative
range of the Genomic DNA ScreenTape
assay (10–100 ng/µL). The dilution
factors for the TapeStation analysis
ranged from 1 to 15.
RNA starting material was diluted 1:10
for quantification with the Qubit RNA
HS assay. The samples were analyzed
undiluted with the RNA ScreenTape
assay for quantification and sample
integrity assessment. For comparison,
the samples were analyzed with the RNA
6000 Nano assay (2100 Bioanalyzer
system).

DNA library preparation
The SureSelectXT protocol1 was used
for whole exome sequencing (WES) as
presented in Figure 1.
The samples were quantified using boththe Genomic DNA ScreenTape assay
and the Qubit dsDNA HS assay. Intact
gDNA was fragmented using the Covaris
instrument according to the SureSelectXT
protocol instructions. The treatment time
was reduced to 240 seconds to increase
the target size. Fragment length andsize distribution of the sheared DNA wasdetermined using the D1000 ScreenTape
assay (4150 TapeStation system) andthe DNA 1000 assay (2100 Bioanalyzer
system). Successfully sheared samples
were subjected to the next step;combining end repair, adenylation, andadapter ligation followed by amplificationand purification (Figure 1). The adapter
ligated samples were again qualified withthe same assays. Successful adapter
ligated samples were then hybridized
and captured, followed by indexing andamplification. After purification, thelibraries were quantified with the Qubit
dsDNA HS assay, then normalized toapproximately 2 ng/µL and quantified
with the High Sensitivity (HS) D1000
ScreenTape assay. The sizes of the final
libraries were automatically determined
during the same step with the HS D1000
ScreenTape assay. Additionally, theA

B

Agilent SureSelectXT

Illumina TruSeq Stranded Total RNA

Genomic DNA

QC

Fragmentation

QC

Total RNA

gDNA ScreenTape

RNA ScreenTape
RNA Nano Assay

D1000 ScreenTape
DNA 1000 Assay

Fragmentationof mRNA

End repair, adenylation, adapter ligation

First and second strand synthesis

Amplification

End repair, adenylation, adapter ligation

Purification

Adapter
Ligated library

QC

Amplification, purification
D1000 ScreenTape

QC

DNA 1000 Assay

Final library

QC

HS D1000 ScreenTape
HS DNA Assay

Sequencing

Hybridization and capture
Indexing and amplification
Purification

Final library

QC

HS D1000 ScreenTape
HS DNA Assay

Sequencing
Figure 1. Overview of whole exome and transcriptome library preparation protocols. A) Workflow of the Agilent SureSelect XT protocol B) Library preparation
steps with the Illumina TruSeq Stranded Total RNA protocol. QC steps were performed with the Agilent 4150 TapeStation system and the ScreenTape assays as
indicated in the blue boxes. Grey boxes refer to the assays used with the Agilent 2100 Bioanalyzer system.

library sizes were assessed with theHigh Sensitivity (HS) DNA assay using
the 2100 Bioanalyzer system. The final
libraries were normalized, pooled, andtransferred to the Illumina HiSeq 2500
sequencer.

RNA library preparation
The TruSeq Stranded Total RNA
protocol2 was used to prepare libraries
for transcriptome sequencing aspresented in Figure 1 with total RNA
as starting material. The integrityof the RNA samples was evaluatedwith the RNA ScreenTape assay(4150 TapeStation system) andcompared to the results of the RNA 6000

Nano assay (2100 Bioanalyzer system).
Both systems automatically provide
quantitative data. The samples wereadditionally quantified after 1:10 dilution
using the Qubit RNA HS assay.
Depending on RNA integrity,
determined with the RNA integrity
number (RIN), the incubation time
for total RNA fragmentation was
modified (RIN > 3.5: 7 minutes;
RIN 2.0–3.5: 2 minutes and RIN < 2.0:
no fragmentation). Fragmented RNA
samples were processed according
to the Illumina TruSeq Stranded Total
RNA protocol with the first and second
strand synthesis, followed by end repair,adenylation, and adapter ligation. After

amplification, the yield of the purified
final libraries was determined with the
Qubit DNA HS assay. The samples werenormalized to approximately 2 ng/µL(dilution factor 10 to 15) and analyzed
on the HS D1000 ScreenTape assay toassess library size and concentration.
The size was also determinedwith the HS DNA assay using the2100 Bioanalyzer system.
All final libraries were pooled and thensequenced with the Illumina HiSeq 2500
sequencer.

3

30 samples of genomic DNA extracted
from genetically engineered mousemodels were manually preparedaccording to the SureSelectXT protocol.

Genomic DNA integrity
Quantity and integrity of gDNA were
evaluated to ascertain that the starting
material was suitable for library
preparation. This first important QC
step was performed with the 4150
TapeStation system and the Genomic
DNA ScreenTape assay. Genomic DNA
quality assessment based on the DNA
integrity number (DIN) was previously
demonstrated.3 The DIN algorithm isincluded in the TapeStation software
providing an objective quality metric forstandardized DNA integrity assessment.
Signal distribution across the sizing
range of the assay is converted into a
numerical value ranging from 10 (intact
DNA) to 1 (degraded DNA). Freshly
extracted gDNA from mouse tissue is
expected to depict high DIN values. The
DIN values of the 30 gDNA samples
ranged from 6.6 to 9.3 (data not shown).
Since high quality could be verified forthe starting material, all 30 samples wereused for library preparation.

The SureSelect protocol is optimized
for the preparation of libraries from3 µg gDNA starting material. The
protocol recommends the Qubit systemfor quantification, which was used
to evaluate gDNA concentration. In
parallel, the samples were quantified
with the 4150 TapeStation systemand the Genomic DNA ScreenTape
assay. DNA samples were normalized
before the analysis with the Genomic
DNA ScreenTape assay. The DNA
quantity determined by both methods
is compared in Figure 2. The
concentration data evaluated with
the Genomic DNA ScreenTape assay
was highly comparable with the Qubit
data exhibiting an R2 correlation valueof 98.5%. This result shows that the
Genomic DNA ScreenTape assay enables
XT

reliable and accurate quantification for
genomic DNA as starting material for
NGS applications.

Characterization of sheared DNA
The first step of the SureSelectXT
protocol is fragmentation of gDNA byshearing with the Covaris ultrasonicator.
The shearing protocol was adjusted
to produce fragments with a larger
target size to allow for sequencing in
2 × 100 bp mode. Optimal shearing in
NGS workflows is typically verified withthe evaluation of the size distribution andelectropherogram pattern of fragmented
DNA samples with the DNA 1000 assay
and the 2100 Bioanalyzer system. In
addition, the samples were analyzed withthe 4150 TapeStation system and theD1000 ScreenTape assay.

900

800
y = 0.9633x
R² = 0.9852
700

Concentration (ng/µL)

DNA library preparation

Genomic DNA quantification

Genomic DNA ScreenTape assay

Results and discussion

600

500

400

300

200

100

0
0

100

200

300

400

500

600

700

800

900

Concentration (ng/µL)
Qubit dsDNA HS assay

Figure 2. Comparison of gDNA concentrations measured with the Agilent Genomic DNA ScreenTape
assay and with the Qubit dsDNA HS assay. The samples were diluted to match the assay ranges. The
displayed concentrations represent the original sample concentrations.

4

A

120
100
80
60
40
20

1,500

700

500

400

300

200

150

Size (bp)
1,
00
Up 0
pe
r

10
0

×103
16

Lo
w

er

100

0
15
50

Due to the modified shearing protocol,
the expected average size range offragmented DNA is about 100 bp larger
than recommended in the protocol(between 200 and 325 bp). The
fragmented DNA samples showed amaximum peak size between 260 and
310 bp on both systems, which verified
optimal shearing. The size of this QC
step can be used as a core value for eachsample to compare with the size after
adapter ligation later in the SureSelectXT
workflow. The sizing results of the 2100
Bioanalyzer and the 4150 TapeStation
systems correlated highly with anaverage deviation of 2.2% (Figure 4).

FU

Sample intensity (normalized FU)

Example electropherograms of sheared
DNA analyzed with the 4150 TapeStationand the 2100 Bioanalyzer systems areshown in Figure 3. All samples displayed
an even size distribution with noundesirable shouldering.

B

14
12
10
8
6
4
2

1,000
1,500

700

500

400

300

200

100

50

25

0

Size (bp)

Figure 3. A fragmented DNA sample from the SureSelect XT workflow showing even size distribution.
A) Sheared DNA analyzed with the Agilent DNA 1000 assay, B) Electropherogram of the same sample
analyzed with the Agilent D1000 ScreenTape assay.

400

DNA 1000 assay
D1000 ScreenTape assay

350

Average size (bp)

300
250
200
150
100
50
0

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

Figure 4. Average size of 30 sheared DNA samples with a region set from 100 to 1000 bp. The average size was determined with the Agilent DNA 1000 assay
and the Agilent D1000 ScreenTape assay.

5

Qualification of precapture libraries
The next steps for sheared DNA in the
SureSelectXT protocol are comprisedof end repair, adenylation, and adapter
ligation followed by amplification.
To verify efficient adapter ligation,
purified adapter ligated DNA samples(precapture samples) were qualified with
the DNA 1000 assay (2100 Bioanalyzer
system) and the D1000 ScreenTape
assay (4150 TapeStation system). The
precapture libraries were analyzed byregion analysis with boundaries set
from 200 to 700 bp. The average region
size ranged from 340 to 420 bp with amaximum peak size at approximately
300 bp. All samples showed an expected
size shift (approximately 30 bp) withregards to the sheared DNA samples,
which is related to adapter ligation.
The average size determined by both
systems was highly comparable
for all 30 samples with an average
deviation of 2.7% (Figure 5). Hence, theaverage region size of libraries can be
confidentially assessed using the region
functionality with the DNA 1000 assay
as well as with the D1000 ScreenTape
assay.

Final library QC
Adapter-ligated samples were processed
according to the SureSelectXT protocol,
including hybridization and capturing,followed by indexing and amplification.
In general, final libraries are equimolar
pooled for sequencing, which requires a
QC step to ensure accurate and precise
evaluation of library size and quantity.
The libraries were analyzed with the HS
DNA assay (2100 Bioanalyzer system)
and with the HS D1000 ScreenTape
assay (4150 TapeStation system).
Electropherogram patterns with a broad
single peak and the absence of artifacts
represent high-quality libraries.4 All
samples depicted high-quality libraries,
example electropherograms are shownin Figure 6.

450

DNA 1000 assay
D1000 ScreenTape assay

400

Average size (bp)

350
300
250
200
150
100
50
0

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

Figure 5. Comparison of the average size of 30 precapture libraries with a region set from 200 to 700 bp. The average size was determined with the Agilent DNA
1000 assay and the Agilent D1000 ScreenTape assay.

6

The maximum peak size ranged from
350 to 400 bp. Due to the increased
fragment size that was aimed during
shearing, the expected average
size ranges from 325 to 400 bp.
To determine the average size formolarity calculation, a region was set
ranging from 200 to 600 bp. The sizing
results of the HS D1000 ScreenTape
assay were compared to the results
determined by the HS DNA assay withthe 2100 Bioanalyzer system. The results
were highly comparable for all samples
with an average deviation of 0.9%
(Figure 7).

A

200

150

100

50

r
pe
Up

60
0

20
0

Lo
w

500

Size (bp)10,380

2,000

1,000

600

500

400

300

200

150

100

er

35

0

Sample intensity (normalized FU)

Both systems provide reliable results for
final library size determination.

FU

B

400

300

200

100

1,000
1,500

700

500

400

300

200

100

50

25

0

Size (bp)

Figure 6. Example electropherograms of end products of the SureSelect XT workflow. A) Finallibrary analyzed with the Agilent HS DNA assay, B) Sample analyzed with the Agilent HS D1000
ScreenTape assay.

450

HS DNA assay
HS D1000 ScreenTape assay

400

Average size (bp)

350
300
250
200
150
100
50
0

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

Figure 7. Sizing comparison of the final libraries generated with the Agilent SureSelect XT workflow. The size was determined by region analysis ranging from 200
to 600 bp. Sizing data was determined with the Agilent HS DNA assay and compared to the Agilent HS D1000 ScreenTape assay.

7

Alacris Theranostics uses the average
size provided by the TapeStation
analysis software and the concentration
determined with the Qubit assay
to determine molarity data. The
concentration data provided by theHS D1000 ScreenTape was comparedto the quantification results attained
from the Qubit dsDNA HS assay(Figure 8). The quantification with
HS D1000 ScreenTape assay was highly
comparable to Qubit results with anaverage deviation of 11.1%.

The electropherogram pattern of DNA
smears and libraries can differ between
the 4150 TapeStation system andthe 2100 Bioanalyzer system due todifferences in technology. Nevertheless,resulting sizing data was highly
comparable between both systems.
Furthermore, the electropherogram
view of either system enabled visualevaluation of an even size distribution
and the absence of artifacts. The
quantification of final libraries with the4150 TapeStation system provided

equivalent values compared to the Qubit
system. Thus, the 4150 TapeStation
system together with the ScreenTape
assay portfolio can be confidentiallyused as a QC tool during library
preparation with the SureSelectXT
protocol.

30
Qubit dsDNA HS assay
HS DNA ScreenTape assay

Concentration (ng/µL)

25

20

15

10

5

0

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

Figure 8. Quantification of SureSelect XT final libraries with the Qubit dsDNA HS assay compared to the Agilent HS D1000 ScreenTape assay. The samples werediluted to match the Agilent HS D1000 ScreenTape assay range. The graph shows the original library concentration.

8

RNA library preparation

Total RNA quantification

Total RNA samples of the same mousemodels used for WES were subjected
to library preparation for transcriptome
analysis with the Illumina TruSeq
Stranded Total RNA protocol (Figure 1).
Final libraries were qualified, pooled, andthen sequenced with an Illumina HiSeq
2500 system.

Total RNA quantification is required
to normalize the starting material
before library preparation. The RNA
samples were quantified with the RNA
ScreenTape assay and comparedto Qubit results (Figure 10). Both10

Total RNA integrity

The RIN algorithm of the TapeStation
software is equivalent to the RIN metricprovided by the RNA assays of the 2100
Bioanalyzer system.5,6 The comparative
analysis of the RNA samples verified
that RIN and RINe are highly correlatingwith a slope of 0.997 and a linear
regression (R2) of 97.9%. Despite thepartial degradation of some samples(RIN < 6), libraries were created fromall RNA samples. The RNA could not be
re-extracted since only very limited tumor
tissue was provided. The Illumina TruSeq
Stranded Total RNA protocol permits theuse of partially degraded RNA as starting
material by modifying the protocol
according to the RNA integrity.

98
RINe (4150 TapeStation system)

RNA samples are often subject todegradation by enzymatical, chemical, orother environmental impacts. Therefore,
RNA integrity analysis is a crucial step
before any downstream application.
In general, Alacris Theranostics
successfully processes RNA starting
material with a RIN above 6 forsequencing. RNA integrity of the starting
material was determined with the RNA
ScreenTape assay (4150 TapeStation
system) and compared to the RNA 6000
Nano assay (2100 Bioanalyzer system)(Figure 9).

systems resulted in similar total RNA
concentrations of the samples with aslope of 1.017 and a linear regression of
97.9% showing high correlation. The two
degraded RNA samples 6 and 7 weretreated as outliers.

y = 0.9973x – 0.1922
R² = 0.9788

7
6
5
4
3
2
1
0
0

1

2

e

3
4
5
6
7
RIN (2100 Bioanalyzer system)

8

9

10

Figure 9. RINe and RIN correlation of 30 mouse total RNA samples analyzed
with the Agilent RNA ScreenTape assay and the Agilent RNA 6000 Nano assay.

Concentration (ng/µL) RNA ScreenTape assay

800

y = 1.0172x + 16.555
R² = 0.9791

700

600

500

400

300

200

100

0

0

100

200

300

400

500

600

700

Concentration (ng/µL) Qubit RNA HS assay

Figure 10. Quantification of 30 mouse total RNA samples with the Agilent RNA
ScreenTape assay and the RNA HS assay (Qubit system). The two outliers inorange refer to samples 6 and 7 which exhibited a high level of degradation.

9

The determined concentration with
the RNA ScreenTape assay of both
samples was approximately 60% lower
compared to the results of the Qubit RNA
HS assay. During gel electrophoresis,
RNA is separated by size and very small
fragments migrate outside the detection
window, thereby decreasing themeasured concentration. Both samples
were processed despite their lack ofquality, however, sequencing results
revealed insufficient performance.

FU
160

Overall, the RNA ScreenTape assay canbe considered as a very useful tool todetermine both integrity and quantity ofRNA starting material for sequencing in asingle analysis step.

0

A

140
120
100
80
60
40

10,380

2,000

1,000

600

500

400

300

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150

100

Size (bp)Sample intensity (normalized FU)

500

pe
r
Up

50
0

B

400

300

200

100

500

1,500

1,000

700

500

400

300

200

100

50

25

0

Sample intensity (normalized FU)

RNA samples were fragmented
according to the Illumina TruSeq
Stranded Total RNA protocol by applying
modified fragmentation protocols
for partially degraded RNA (RIN < 6).
Fragmented RNA samples were
processed through the first and second
strand synthesis, followed by end repair,adenylation and adapter ligation, thenamplification. The purified final libraries
were characterized using the HS D1000
ScreenTape assay (4150 TapeStation
system) in addition to the HS DNA assay(2100 Bioanalyzer system). Figure 11
shows example electropherograms forboth systems, while Figure 12 compares
the sizing results. The electropherogramsof all samples reflected high-quality
patterns showing a single broad librarywithout any additional peaks. However,
the two degraded RNA samples 6 and 7
depicted an anomalous library pattern, as
shown in Figure 11C.

Lo

Characterization of final libraries

20
0

w

er

35

20

Size (bp)C
Sample 6
Sample 7
Sample 8

400

300

200

100

1,500

1,000

700

500

400

300

200

100

50

25

0

Size (bp)

Figure 11. Comparison of electropherogram patterns of final libraries prepared from RNA. A) Final library
analyzed with the Agilent DNA 1000 assay. B) Electropherogram of the same sample obtained with theAgilent D1000 ScreenTape assay. C) Overlay of a high-quality library (sample 8) and two libraries fromdegraded starting material (samples 6 and 7) analyzed with the D1000 ScreenTape assay.

10

The average size of the final libraries wasdetermined by setting an analysis region
from 200 to 500 bp and ranged from 300
to 390 bp. This matched the expected
average size range between 250 and
400 bp. The sizing results of the HS
D1000 ScreenTape assay were similar
to the results determined by the HS DNA
assay (2100 Bioanalyzer system), withan average deviation of 1.2%.

Library yield was automaticallydetermined with the HS D1000
ScreenTape assay and was compared
to results provided by the Qubit
dsDNA HS assay (Figure 13). The
libraries were diluted to the HS D1000
ScreenTape assay quantitative range.
The data reflects the original library
concentrations before dilution. The final
library concentration determined byboth systems showed a high correlation,with an average deviation of 12.9%.
This result reveals that the HS D1000
ScreenTape assay can be used for libraryyield determination equivalent to the
Qubit system. During the same analysis
step, library size can be determined withequal results compared to the HS DNA
assay of the 2100 Bioanalyzer system.

450

HS DNA assay

400

HS D1000 ScreenTape
assay

Average size (bp)

350
300
250
200
150
100
50
0

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

Figure 12. Sizing results for 30 final libraries of mouse RNA samples analyzed with the Agilent HS D1000 ScreenTape assay and the Agilent HS DNA assay.

20

Qubit dsDNA HS assay

18

HS D1000 ScreenTape
assay

Concentration (ng/µL)

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8
6
4
2
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2

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

Figure 13. Quantification of 30 final libraries evaluated with the HS D1000 ScreenTape assay compared to data determined with the Qubit system. The data
reflects the original library concentrations before dilution.

11

The final libraries depicted high yield andquality and were normalized, pooled,and sequenced. Two RNA samples witha high level of degradation showed low
RIN and RINe numbers and deviating
quantities when comparing Qubit and4150 TapeStation system data. Libraries
generated from these samples resulted
in lower library yield and a different
electrophoresis pattern and finally
showed low library complexity.

Conclusion
Alacris Theranostics successfully
evaluated the 4150 TapeStation systemwith the ScreenTape assay portfolio tomonitor exome and transcriptome library
preparation for systems biology omics
analysis. 30 DNA and RNA samples wereextracted from genetically engineeredmouse models and sequenced as partof the CanPathPro project. The 4150
TapeStation system was comparedwith the 2100 Bioanalyzer and Qubit
systems by performing quality control
steps on the platforms in parallel. It wasdemonstrated that the 4150 TapeStation
system is a reliable tool for sizing andquantification at any step of library
preparation for exome and transcriptome
sequencing. It can be used to qualify
sheared DNA, precapture libraries,
and final libraries with sizing results

www.agilent.com/genomics/
tapestation
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, June 5, 20195994-0946EN

equivalent to the 2100 Bioanalyzer
system. Quantitative data of all employed
ScreenTape assays highly correlatedwith Qubit results. In addition, the4150 TapeStation system consistently
delivered excellent RNA characterization
for quality assessment. It provides the
RNA integrity number equivalent (RINe),which is highly comparable to the well
accepted RIN algorithm of the 2100
Bioanalyzer system. Poor sequencingof two degraded RNA samples could
be anticipated by the interpretationof QC data assessed with the 4150
TapeStation system. The ready-to-use
ScreenTape devices simplify the quality
control steps that are part of the library
preparation workflow, and significantly
reduce hands-on-time compared to the2100 Bioanalyzer system. Moreover,the 4150 TapeStation system enables
reliable quality and quantity assessmentof genomic DNA with the quality metric
DIN. The 4150 TapeStation system
can load any sample number from 1 to
16 samples per run without wasting
consumables, thus providing a constantcost-per-sample. Together with theeasy switch between different assays,
this gives the user high flexibility toeasily accommodate to project‑driven
throughput.

References
1.

Agilent SureSelectXT Target
Enrichment System for Illumina
Paired-End Multiplexed Sequencing,
Agilent Technologies User Manual,publication number G7530-90000,
2017.

2.

TruSeq Stranded Total RNA
Reference Guide, Illumina document
no. 1000000040499 v00, 2017.

3.

Quality Control in Illumina
Sequencing Workflows Using
the TapeStation System, Agilent
Technologies Application Note,publication number 5994-0327EN,
2018.

4.

Sample Quality Control in Agilent
NGS Solutions, Agilent Technologies
Application Note, publication number
5994-0127EN, 2018.

5.

Performance of the Agilent RNA
ScreenTape and the High Sensitivity
RNA ScreenTape Assay for the
Agilent 2200 TapeStation System,
Agilent Technologies Technical
Overview, publication number
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