Use of Agilent 4200 TapeStation
system for external quality
assurance of genomic DNA and
cell-free DNA
Genomic DNA ScreenTape®
Authors
Page 1 of 69
TapeStation Gel Images
Wim Ammerlaan(1), Olga Kofanova(1), Rainer Nitsche(2), Fay Betsou(1) and the ISBER Proficiency Testing Advisory Group
(1)
Integrated BioBank of Luxembourg (IBBL), 6 rue Nicolas Ernest Barblé, 1210 Luxembourg, Luxembourg
(2)
Agilent Technologies, Waldbronn, Germany (This work was part-funded by Agilent Technologies and IMI grant no. 115749.)
Filename: 2016-11-16 - 13.11.17.GDNA
Gel
GelImages
Images - gDNA from BD Paxgene Blood DNA tubes
Background
EQAs is mandatory for quality assurance of the wide variety of DNA extraction methods. DNA quality
assessment by integrity and molecular size estimation can be achieved using conventional gel or automatic
microfluidic electrophoresis systems (e.g Agilent 2100 Bioanalyzer system)
Here we evaluated the Agilent 4200TapeStation system, in the context of the ISBER-endorsed “Proficiency Testing (PT)”
program organised by IBBL, for :
• Genomic DNA (gDNA) extraction methods from
1.Whole blood (BD Paxgene Blood DNA tubes);
Default image (Contrast 50%), Image is Scaled to Sample
2.Snap frozen tissue;
3.Formalin-Fixed Paraffin-Embedded (FFPE) cell pellet.
• Cell-free DNA (cfDNA) extraction methods from plasma isolated from whole blood collected in PaxGene cfDNA tubes,
Fig.1 DNA extracted from blood collected in Paxgene DNA tubes
analysed with the Agilent genomic DNA ScreenTape assay. Lanes L1
and L2 are DNA ladders. Note the DNA degradation pattern in most
samples.
spiked-in with double stranded DNA ultramers of 200bp covering several standard cancer mutations.
4200 TapeStation System
Gel Images – gDNA from snap frozen tissue
• Automated microfluidic electrophoresis system:
• 96 well plate or 2x8 well strip sample loading format;
• Individual ScreenTape devices consisting of 16 lanes, i.e. 15 samples and 1 ladder;
Default image (Contrast 50%), Image is Scaled to Sample
• Multiple ScreenTape devices loading for maximum 96 samples run in walk away modus;
• 1 μl sample volume needed per run in concentration of 10-100 ng/μl for genomic DNA and 10-1000 pg/μl for High
Sensitivity D5000 ScreenTape assays.
Genomic DNA ScreenTape®
Page 1D5000
of 30 ScreenTape®
High Sensitivity
Page 1
Fig.2 DNA extracted from fresh frozen tissue analysed with the Agilent
genomic DNA ScreenTape assay. Lanes L1 and L2 are DNA ladders.
Filename: 2016-11-16 - 17.41.20.GDNA
Filename: 2016-11-17 - 17.19.09.HSD5000
Gel Images - cfDNA
Gel
GelImages
Images - gDNA from FFPE cell pellet
Gel Image
Default image (Contrast 50%), Image is Scaled to Sample
TapeStation Analysis Software A.02.01 SR1
Default image (Contrast 100%), Image is Scaled to view larger Molecular Weight range
Fig.3 DNA extracted from fresh frozen tissue analysed with
the Agilent genomic DNA ScreenTape assay. Lanes L1 and L2
are DNA ladders. Note the DNA smear in most samples and
the lower DIN score compared to the DNA from blood.
Fig.4Info
cfDNA extracted from plasma analyqed with the
Sample
Agilent
ScreenTape assay.
L1 is DNA
Well HS DNA5000
Conc. [pg/µl]
Sample Lane
Description
A1
2830
Ladder
ladder.
of IBBL_PT2016_cfDNA16_L018
DIN score cannot be applied to
B1 Note the absence
14800
C1
IBBL_PT2016_cfDNA16_L124
cfDNA.
D1
4500
IBBL_PT2016_cfDNA16_L083
For gDNA:
For cfDNA:
A1
2.7
8.21
IBBL PT
2016_DNAFFC16_L018
B1
3.6
4.42
IBBL PT
2016_DNAFFC16_L037
IBBL PTDNA tubes
Fig.5 All C1Results - gDNA
Blood
5.1 from BD Paxgene
15.3
D1
5.4
13.5
E1
5.7
19.1
L2
-
74.2
F1
5.7
17.3
G1
5.4
26.3
H1
5.1
25.2
2016_DNAFFC16_L146
IBBL PT
2016_DNAFFC16_L035
IBBL PT
2016_DNAFFC16_L119
Ladder
IBBL PT
2016_DNAFFC16_L256
IBBL PT
2016_DNAFFC16_L108
IBBL PT
2016_DNAFFC16_L186
Alert
Observations
Ladder
Sample concentration
outside recommended
range
Sample concentration
outside functional range
for DIN
Fig.6 All Results - gDNA from snap frozen tissue
All results
No. of laboratories:
No. of outlier values:
Mean:
Proficiency Testing Standard Deviation:
Assigned value:
Assigned value:
Ladder
80.0
• FFPE cell pellets DIN 4.89 (STDEV 1.05) (Fig.3, 7).
• 81-89% integrated area corresponding to the 200bp peak of the spike-in;
Median:
Minimal value:
Maximal value:
Range:
85.80
71.80
89.30
17.50
B
Laboratory
PROLab Plus
Fig.7 All Results - gDNA from FFPE cell pellets
TapeStation Analysis Software A.02.01 SR1
© Agilent Technologies, Inc. 2016
Fig.8
All Results - cfDNA
Magnetic-bead based Results
Generated:
16-Nov-2016
TapeStation
Analysis Software A.02.01 SR1
© Agilent Technologies, Inc. 2016
No. of laboratories:
No. of outlier values:
Mean:
Proficiency Testing Standard Deviation:
Assigned value:
Assigned value:
• Contamination by cell gDNA was evident in samples from some participants;
%
• TapeStation HS D5000 kit outperformed the Bioanalyzer DNA1000 kit and the TapeStation HS D1000 kit.
Median:
Minimal value:
Maximal value:
Range:
84.40
71.80
89.30
17.50
100
L214
The 4200 TapeStation system is well suited for the purpose of quality control of DNA samples extracted from clinical material;
4
0
82.48 %
7.95 % (Empirical value)
82.48 % (Empirical value)
82.48 %
Generated: 07-Dec-
M
ea
n
For cfDNA, the following results were obtained from all participants (Fig.4, 8) :
6
1
86.05 %
2.86 % (Empirical value)
86.05 % (Empirical value)
86.05 %
M
ea
n
Sample Description
it is high throughput and user-friendly. Particularly in
Laboratory
PROLab Plus
the case of cfDNA, the 4200 TapeStation system allows simultaneous assessment of the size of the extracted cfDNA and of any
Silica-membrane based Results
potential contamination by white blood cell DNA.
No. of laboratories:
No. of outlier values:
Mean:
Proficiency Testing Standard Deviation:
Assigned value:
Assigned value:
Median:
Minimal value:
Maximal value:
Range:
85.80
85.00
87.40
2.40
90
85
L253
%
Conclusions
3
0
86.07 %
1.22 % (Empirical value)
86.07 % (Empirical value)
86.07 %
M
ea
n
• Snap frozen tissue DIN 5.67 (STDEV 1.57) (Fig.2, 6);
Ladder
L248
• Stabilised blood DIN 6.7 (STDEV 0.75) (Fig.1, 5);
71.6
L248
For gDNA, average DIN score from all participants in our external Proficiency Testing programme:
Conc. [ng/µl]
-
L083
Results
DIN
L1
The 4200 TapeStation sytem is more informative than the 2100 Bioanalyzer system, and is well suited for the purpose of quality control of DNA samples extracted
L261
corresponding to the size of the spiked-in dsDNA ultramers.
Well
L261
• We evaluated the extracted cfDNA quality from each participant based on the % integrated area around the 200 bp peak,
Marker(s) not detected
Biorepository proficiency testing
for the quality control
of biospecimens
Sample Info
L214
Bioanalyzer DNA 1000 kit to determine the best detection system;
Observations
Ladder
Default image (Contrast 50%), Image is Scaled to Sample
%
• We ran cfDNA with the 4200 TapeStation High Sensitivity D1000 and High Sensitivity D5000 kit as well as the 2100
L083
• We measured DIN scores, ranging from 1 (DNA completely degraded) to 10 (DNA completely intact).
PT Results
1590
119
5.37
6.59
Alert
IBBL_PT2016_cfDNA16_L214
IBBL_PT2016_cfDNA16_L253
IBBL_PT2016_cfDNA16_L216
IBBL_PT2016_cfDNA16_L248
DNA_QC_A_2015_1_dil10000
IBBL_PT2016_cfDNA16_mix100
IBBL_PT2016_cfDNA16_mix1000
IBBL_PT2016_cfDNA16_mix10000
IBBL_PT2016_cfDNA16_mix100000
IBBL_PT2016_cfDNA16_mix1000000
IBBL_PT2016_cfDNA16_mix10000000
NTC
L124
sample run with the Genomic DNA ScreenTape assay;
14900
396
23900
7090
L124
• We evaluated the extracted DNA quality from each participant, by determining a DIN (DNA Integrity Number score for each
E1
F1
G1
H1
A2
B2
C2
D2
E2
F2
G2
H2
L253
Methods
Default image (Contrast 50%), Image is Scaled to Sample
L018
ScreenTape Devices for DNA and RNA Assays
Generated: 16-Nov-2016
L018
Agilent 4200 TapeStation Instrument
© Agilent Technologies, Inc. 2016
Laboratory
PROLab Plus
from clinical material; it is high throughput and user-friendly and introduces DIN values, a quality label for DNA samples. Particularly in the case of cfDNA, the 4200
TapeStation system allows simultaneous assessment of the size of the extracted cfDNA and of any potential contamination by white blood cell DNA.
Final
CFDNA16
without highlighting
Prepared on: 14 March 2017
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