Use of Agilent 4200 TapeStationsystem for external quality assurance of genomic DNA and cell-free DNA

Background

4200 TapeStation System

Methods

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);

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,

spiked-in with double stranded DNA ultramers of 200bp covering several standard cancer mutations.

• 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;

• 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.

For gDNA:

• We evaluated the extracted DNA quality from each participant, by determining a DIN (DNA Integrity Number score for each

sample run with the Genomic DNA ScreenTape assay;

• We measured DIN scores, ranging from 1 (DNA completely degraded) to 10 (DNA completely intact).

For cfDNA:

• We ran cfDNA with the 4200 TapeStation High Sensitivity D1000 and High Sensitivity D5000 kit as well as the 2100

Bioanalyzer DNA 1000 kit to determine the best detection system;

• We evaluated the extracted cfDNA quality from each participant based on the % integrated area around the 200 bp peak,

corresponding to the size of the spiked-in dsDNA ultramers.

AuthorsWim 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.)

TapeStation Gel Images

Agilent 4200 TapeStation Instrument ScreenTape Devices for DNA and RNA Assays

Filename: 2016-11-16 - 13.11.17.GDNA

Gel Images

Default image (Contrast 50%), Image is Scaled to Sample

Default image (Contrast 50%), Image is Scaled to Sample

Default image (Contrast 50%), Image is Scaled to Sample

Genomic DNA ScreenTape® Page 1 of 69

TapeStation Analysis Software A.02.01 SR1 © Agilent Technologies, Inc. 2016 Generated: 16-Nov-2016

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.

Gel Images - gDNA from BD Paxgene Blood DNA tubes

Filename: 2016-11-17 - 17.19.09.HSD5000

Gel Image

Default image (Contrast 100%), Image is Scaled to viewlarger Molecular Weight range

Sample Info

Well Conc. [pg/µl] Sample Description Alert Observations

A1 2830 Ladder Ladder

B1 14800 IBBL_PT2016_cfDNA16_L018

C1 IBBL_PT2016_cfDNA16_L124 Marker(s) not detected

D1 4500 IBBL_PT2016_cfDNA16_L083

E1 14900 IBBL_PT2016_cfDNA16_L214

F1 396 IBBL_PT2016_cfDNA16_L253

G1 23900 IBBL_PT2016_cfDNA16_L216

H1 7090 IBBL_PT2016_cfDNA16_L248

A2 DNA_QC_A_2015_1_dil10000

B2 1590 IBBL_PT2016_cfDNA16_mix100

C2 119 IBBL_PT2016_cfDNA16_mix1000

D2 5.37 IBBL_PT2016_cfDNA16_mix10000

E2 6.59 IBBL_PT2016_cfDNA16_mix100000

F2 IBBL_PT2016_cfDNA16_mix1000000

G2 IBBL_PT2016_cfDNA16_mix10000000

H2 NTC

High Sensitivity D5000 ScreenTape® Page 1 of 19

TapeStation Analysis Software A.02.01 SR1 © Agilent Technologies, Inc. 2016 Generated: 07-Dec-2016

Fig.4 cfDNA extracted from plasma analyqed with the

Agilent HS DNA5000 ScreenTape assay. Lane L1 is DNA

ladder. Note the absence of DIN score cannot be applied to

cfDNA.

Gel Images - cfDNAFilename: 2016-11-16 - 17.41.20.GDNA

Gel Images

Default image (Contrast 50%), Image is Scaled to Sample

Default image (Contrast 50%), Image is Scaled to Sample

Sample Info

Well DIN Conc. [ng/µl] Sample Description Alert Observations

L1 - 71.6 Ladder Ladder

A1 2.7 8.21IBBL PT2016_DNAFFC16_L018

Sample concentrationoutside recommendedrange

B1 3.6 4.42IBBL PT2016_DNAFFC16_L037

Sample concentrationoutside functional rangefor DIN

C1 5.1 15.3IBBL PT2016_DNAFFC16_L146

D1 5.4 13.5IBBL PT2016_DNAFFC16_L035

E1 5.7 19.1IBBL PT2016_DNAFFC16_L119

L2 - 74.2 Ladder Ladder

F1 5.7 17.3IBBL PT2016_DNAFFC16_L256

G1 5.4 26.3IBBL PT2016_DNAFFC16_L108

H1 5.1 25.2IBBL PT2016_DNAFFC16_L186

Genomic DNA ScreenTape® Page 1 of 30

TapeStation Analysis Software A.02.01 SR1 © Agilent Technologies, Inc. 2016 Generated: 16-Nov-2016

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.

Gel Images - gDNA from FFPE cell pellet

Fig.7 All Results - gDNA from FFPE cell pellets

PT Results

Fig.5 All Results - gDNA from BD Paxgene Blood DNA tubes Fig.6 All Results - gDNA from snap frozen tissue

Fig.8 All Results - cfDNA

Final CFDNA16 without highlighting Prepared on: 14 March 2017 Page 12 of 19

Biorepository proficiency testingfor the quality control

of biospecimens

All results

PROLab Plus

Laboratory

L214

L018

L253

L124

L261

L083

L248

% 80.0

No. of laboratories: 6No. of outlier values: 1Mean: 86.05 %Proficiency Testing Standard Deviation: 2.86 % (Empirical value)Assigned value: 86.05 % (Empirical value)Assigned value: 86.05 %

Median: 85.80Minimal value: 71.80Maximal value: 89.30Range: 17.50

Mean

B

Magnetic-bead based Results

PROLab Plus

Laboratory

L214

L018

L083

L248

%

100

No. of laboratories: 4No. of outlier values: 0Mean: 82.48 %Proficiency Testing Standard Deviation: 7.95 % (Empirical value)Assigned value: 82.48 % (Empirical value)Assigned value: 82.48 %

Median: 84.40Minimal value: 71.80Maximal value: 89.30Range: 17.50

Mean

Silica-membrane based Results

PROLab Plus

Laboratory

L253

L124

L261

%

9085

No. of laboratories: 3No. of outlier values: 0Mean: 86.07 %Proficiency Testing Standard Deviation: 1.22 % (Empirical value)Assigned value: 86.07 % (Empirical value)Assigned value: 86.07 %

Median: 85.80Minimal value: 85.00Maximal value: 87.40Range: 2.40

Mean

Fig.2 DNA extracted from fresh frozen tissue analysed with the Agilent

genomic DNA ScreenTape assay. Lanes L1 and L2 are DNA ladders.

Gel Images – gDNA from snap frozen tissue

ResultsFor gDNA, average DIN score from all participants in our external Proficiency Testing programme:

• Stabilised blood DIN 6.7 (STDEV 0.75) (Fig.1, 5);

• Snap frozen tissue DIN 5.67 (STDEV 1.57) (Fig.2, 6);

• FFPE cell pellets DIN 4.89 (STDEV 1.05) (Fig.3, 7).

For cfDNA, the following results were obtained from all participants (Fig.4, 8) :

• 81-89% integrated area corresponding to the 200bp peak of the spike-in;

• 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.

The 4200 TapeStation system is well suited for the purpose of quality control of DNA samples extracted from clinical material;

it is high throughput and user-friendly. 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.

ConclusionsThe 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

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.

Agilent Publication Number 5991-8605EN