Agilent 2200 TapeStation DNA Analysis Technical Overview

Good Measurement Practices for

DNA Analysis with the Agilent

2200 TapeStation System

Technical Overview

Introduction

The Agilent 2200 TapeStation system is a complete solution for automated, fast, and reliable electrophoresis. It is made up of three elements: the ScreenTape consumable, the 2200 TapeStation instrument, and the analysis software. The 2200 TapeStation system is straightforward to use; simply place the sample tubes and ScreenTape consumable in the instrument and let it load, separate, image, analyze, and present the results in approximately 1–2 minutes per sample.

The 2200 TapeStation system offers scalable throughput and rapid results, making it an ideal solution for quality control of biological samples in next-generation sequencing

(NGS), microarray, and quantitative PCR workfl ows. During assay preparation, the instructions regarding reagent preparation and instrument maintenance must be strictly followed. Important technical points are described in the TapeStation User Manual 1 and other supporting documentation. This Technical Overview describes techniques for ensuring reliable quantifi cation and sizing results using DNA assays on the

2200 TapeStation system.

Experimental

Materials

The 2200 TapeStation system

(G2964AA/G2965AA), D1000 ScreenTape and Reagents (5067-5582 and 5067-5583), and Genomic DNA ScreenTape and reagents (5067-5365 and 5067-5366), were obtained from Agilent Technologies

(Waldbronn, Germany). The IKA MS 3 basic vortexer with PCR plate adapter

(4674100) was purchased from

IKA GmbH & Co. KG (Staufen, Germany www.ika.com).

Quantifi cation

Acquiring DNA concentration data is essential for sample QC. To ensure accurate quantifi cation from the

2200 TapeStation system and its DNA

Assays (D1000, and High Sensitivity

D1000), it is important to note the following:

Sample mixing

In D1000 and High Sensitivity D1000 assays, the protocol states that sample and sample buffer are vortex-mixed using the IKA vortexer and adaptor at 2,000 rpm for 1 minute to ensure proper mixing.

After vortex mixing, it is recommended that the samples be collected at the bottom of the tube by brief centrifugation.

The effects of insuffi cient mixing were investigated using D1000 ScreenTape and reagents, and results are presented in

Figures 1 and 2, as well as Table 1.

It is clearly shown that poor mixing dramatically affects the reported sample concentration, and that following the recommended procedure is the best way to attain accurate quantifi cation results.

A

1,000

800

Vortex mixing

Pipette

Mixing

No mixing

600

400

200

0

120

100

80

60

40

20

Size (bp)

B

[bp]

1,500

1,000

700

500

300

100

25

Figure 1. A) shows the electropherogram and B) the gel image of the ScreenTape mixing tests. In both panels the green trace shows the recommended protocol of vortex mixing using the IKA vortexer and adaptor at 2,000 rpm for 1 minute followed by brief centrifugation. Blue shows results for pipette mixing only. Red shows the effect of no mixing. Images were taken from the Agilent TapeStation Analysis

Software.

0

Vortex mixing then spin down

Pipette mixing only No mixing

Figure 2. Chart of reported concentrations for the ScreenTape mixing tests, using the D1000 Assay.

Concentrations are expressed as a percentage of the theoretical for the three mixing methods. As above, the green bar represents the recommended protocol of vortex mixing using the IKA vortexer and adaptor at 2,000 rpm for 1 minute followed by brief centrifugation; blue - pipette mixing only; red - no mixing.

Table 1. ScreenTape Reagent mixing tests. Quantifi cation values obtained from the TapeStation Analysis

Software and the Agilent D1000 ScreenTape Assay when using the correct protocol (vortex mix followed by brief centrifugation) and two incorrect mixing protocols (pipette mixing only and no mixing) as illustrated in Figure 2.

Measured concentration

(ng/µL)

Vortex mixing then centrifugation 68.5

Pipette mixing only 21.2

No mixing 6.5

Theoretical concentration

(ng/µL)

70

70

70

2

Peak integration

For D1000 and High Sensitivity D1000

ScreenTape, the concentration values are calculated using the area of the sample peak compared to the known concentration of the top marker. It is therefore essential that all peaks are integrated correctly in the TapeStation

Analysis Software. Ensure that both the upper marker and sample peaks are properly integrated by manually adjusting the peak when necessary. Figure 3 shows examples of correct marker peak integration.

Figure 4 demonstrates the effect of peak integration on the quantitative results. Incorrect peak integration can signifi cantly bias the determined DNA sample concentration.

Magnification of the chart on the left

1

0

3

2

×10 3

6

5

4

Lower 258 Upper

Size (bp)

Figure 3. Correct upper marker integration for the 2200 TapeStation DNA Assays. Figure shows D1000

Assay.

2

1

0

4

3

A

×10 3

6

5

Lower

Correct peak integration concentration: 41.2 ng/µL

258 Upper

Size (bp)

2

1

4

3

0

B

×10

6

3

5

Lower

Incorrect peak integration concentration: 14.9 ng/µL

258 Upper

Figure 4. Example of correct (A) and incorrect (B) sample peak integration, and their effect on reported sample concentration.

Size (bp)

NOTE: NGS libraries should be quantifi ed using the region mode of the TapeStation Analysis software.

Please see the section on Peak maxima versus average molecular weight sizing for further details.

3

Use the correct protocol

Each ScreenTape type is designed for use with its corresponding Reagent kit. It is important that the correct reagents are used with the selected ScreenTape. It is also important that the correct sample preparation protocol 2,3 is followed exactly, using the correct volumes of sample and sample buffer as seen in the Figure 5.

Any variations in the volumes shown can adversely affect the quantifi cation results generated from the system.

In addition, it is important to choose the correct assay based on the concentration of the sample. Using sample concentrations outside the specifi ed quantitative ranges (as detailed in Table 2) will lead to inaccurate quantifi cation.

2200 TapeStation foil cover

(p/n 5067-5154) to prevent the sample from leaving the plate during vortexing.

After vortexing, use an appropriate centrifuge for either 96-well plates, or

8-way strips to ensure that all of the samples are at the bottom of the tube before placing in the 2200 TapeStation.

A Agilent D1000 ScreenTape assay

1 µL DNA or

Ladder

3 µL

Sample Buffer

CAUTION: To avoid damage to the

2200 TapeStation instrument and to ensure correct results, use only the recommended consumables and reagents with the 2200 TapeStation system as listed in the TapeStation

User Manual 1 . The use of 96-well plate covers from other suppliers could result in a failure to pierce the fi lm potentially leading to an instrument crash.

Use the correct tools for the job

Use pipettes that are calibrated and suffi cient for the volume to pipette. A

20-µL pipette will not be as accurate at pipetting 1–2 µL as a 2-µL or 10-µL pipette. Ensuring correct pipetting technique assists in ensuring that the volumes used in each assay are precise, and that the concentrations can be calculated correctly.

Use a vortexer designed for mixing 8-way tube strips, or 96-well plates. TapeStation systems are supplied with an optional IKA

MS3 vortexer, which includes a 96-well plate adaptor suitable for both 96-well plates and 8-way strips. This vortexer is recommended for use with the D1000 and High Sensitivity D1000 ScreenTape assays. For all other assays, please consult the assay Quick Guide for mixing recommendations.

It is recommended that TapeStation users obtain the IKA MS3 vortexer for best results. This vortexer can only be obtained directly from IKA (www.ika.com) by quoting the part number 4674100.

Agilent Technologies, Inc. will not sell these parts separately. If an IKA MS3 vortexer is not available, ensure thorough mixing by vortex for 10 seconds on maximum speed.

With a 96-well plate, use the

Vortex 1 minute

Spin down

B Agilent High Sensitivity D1000 and High Sensitivity D5000 ScreenTape assays

2 µL DNA or

Ladder

2 µL

Sample Buffer

Vortex 1 minute

Spin down

Figure 5. Agilent D1000 ScreenTape Assay (A) and High Sensitivity D1000 ScreenTape Assay (B) sample preparation protocols.

Table 2. Specifi ed quantitative ranges for the Agilent D1000 and High Sensitivity D1000 ScreenTape

Assays.

Quantitative range

Agilent D1000 ScreenTape 0.1 ng/µL–50 ng/µL

Agilent High Sensitivity D1000 ScreenTape 10 pg/µL–1,000 pg/µL

4

TapeStation analysis in the Agilent

SureSelect workfl ow

The 2200 TapeStation system has been verifi ed for use within the Agilent

SureSelect protocol. However, tests have shown that the purifi cation step, where DNA libraries are purifi ed using

AMPure XP beads, can have an effect on quantifi cation. It is postulated that carryover of the beads can cause issues, as the beads could be retained at the top of the separation matrix and contribute to the area of the upper marker (Figure 6).

This causes a lower relative reported value for sample concentration. The phenomenon can be identifi ed when there is a visible signal above the upper marker, as seen in Figure 6. A similar profi le can also occur when overamplifi cation of samples causes the signal to run concurrently with the upper marker.

This artifact can be avoided by increasing the time for which the samples are incubated on the magnetic plate to

10 minutes, thereby removing a higher percentage of the beads.

Sizing

Peak maxima versus average molecular weight sizing

The TapeStation analysis software contains options to display both an

Electropherogram view and a Region view. The Region view is designed for analyzing samples that appear as a smear (for example, NGS libraries) and gives slightly different information to that displayed in the electropherogram view (Figure 7). With this in mind, it is important to use the correct function for your samples.

200

150

100

50

Upper

designed for use with discrete peaks, and the default size reported is that of the highest point of the peak. whole smear or region, and reports size as that of the center of the regions’ mass. This gives the user an idea of the distribution of sizes within that sample.

0

Figure 6. Enlarged image of the upper marker showing additional signal from AMPure beads.

Lower

Electropherogram view

179 Upper Lower 70

Region view

700 Upper

Size (bp)

Size: 179 bp

Size (bp)

Average size: 232 bp

Figure 7. The sizing data obtained in Electropherogram and Region views of the Agilent TapeStation

Analysis Software.

5

Identifying the correct markers

It is important to ensure that the correct upper and lower markers are assigned in the TapeStation analysis software. The markers are used within the software as internal references in order to determine the molecular weight of each sample peak. Incorrect identifi cation can lead to miscalculations in the reported sizing values.

A

Figure 8 shows an example of incorrect lower marker identifi cation next to the corrected fi le. In each of the images, lanes 2 and 3 are the same sample at different concentrations. As seen in

Figure 8A, incorrect identifi cation of the lower marker has caused misalignment of the sample; therefore all the fragments are reported with incorrect sizes.

Manually assigning the correct lower marker (Figure 8B) provides accurate sizing information.

B

Flicking the ScreenTape

Due to the nature of the ScreenTape consumables, bubbles can form in the buffer chamber. If bubbles form at the gel/buffer interface, a loss of performance can be observed in that lane.

It is important therefore always to ‘fl ick’ the ScreenTape before placing it into the

2200 TapeStation instrument to move the bubble to the top of the chamber where it will no longer affect sample loading

(Figure 9).

X Incorrectly assigned as the lower marker Correctly assigned as the lower marker

Figure 8. Screenshots of the Agilent TapeStation Analysis Software, showing A) incorrect lower marker identifi cation in the middle lane of the gel image and

B) corrected lower marker in the middle lane. The lower marker is always highlighted with green in the gel image.

X

Figure 9. Flicking the ScreenTape consumable removes bubbles from the gel interface.

6

Bubbles at the gel interface can affect the performance of the run since the bands appear smudged or diagonal (Figure 10).

In turn, this can affect how the bands are identifi ed in the TapeStation Analysis

Software.

[bp]

A1 (L) A2

X

1,500

1,000

700

500

300

B2

X

100

25

Figure 10. The effect of bubbles at the gel interface on the DNA separation profi le

(lanes A2 and B2).

Molarity

In the TapeStation Analysis Software, molarity is determined from both size and quantity. Ensure that good measurement practices for sizing and quantifi cation have been followed to ensure accurate molarity values.

Genomic DNA Assay

When using the Genomic DNA Assay, it is important to note the following recommendations to ensure correct sizing.

Equilibrate reagents to room temperature

Genomic DNA Reagents must be equilibrated to room temperature for

30 minutes before use. Failure to do so can affect sizing results as cold reagents will overestimate the size of genomic

DNA samples (Table 3).

Table 3. The effect of room temperature (RT) equilibrated Genomic DNA reagents as well as cold reagents on the sizing accuracy of the Genomic DNA ScreenTape assay.

MW

Accuracy

Expected

17,000 bp

–

Reagents at RT

18,867 bp

+ 11 %

Reagents at 4 °C

24,369 bp

+ 43 %

Always use fresh genomic

DNA ladder

When using the Genomic DNA

ScreenTape Assay, it is important that the

Genomic DNA Ladder is freshly prepared for each run. For tube strips or 96-well plate, the ladder must always be the fi rst well selected to run. Failure to do so will affect the sizing results obtained from the assay.

It is also important to note that there is no Genomic DNA software saved ladder in the TapeStation Analysis Software.

The effect of shaking the genomic

DNA ladder vial

The Genomic DNA Ladder must only be vortex mixed as shaking the ladder vial can degrade the top fragment (Figure 11).

Therefore, before pipetting the ladder into the TapeStation approved tube strips or

96-well plate, the ladder vial should be handled carefully, then gently vortexed for 5 seconds to maintain accurate performance.

To minimize shaking during transit, the

Genomic DNA reagents are shipped frozen, on dry ice. Once received, these should be kept at 2–8 °C in the refrigerator.

700

600

500

400

300

200

100

0

A

Lo w er

250 40

0

A1:Ladder

600 900 1,

20

0

1,

50

0

2,

00

0 0

2,50 3,

00

0

4,

00

0

7,

00

0

15

,0

00

48

,5

00

Size (bp)

48,500

15,000

7,000

4,000

3,000

2,500

2,000

1,500

1,200

900

600

400

250

100 bp A1 (L)

600

500

400

300

200

100

0

B

Lo w er

250 40

0

A1:Ladder

600 900 1,

20

0

1,

50

0

2,

00

0 0

2,50 3,

00

0

4,

00

0

7,

00

0

15

,0

00 bp A1 (L)

!

Size (bp)

15,000

7,000

4,000

3,000

2,500

2,000

1,500

1,200

900

600

400

250

100

Figure 11. The effect of shaking the Genomic DNA Ladder. A) Genomic DNA Ladder has been vortex mixed for 5 seconds prior to analysis on the Genomic DNA

ScreenTape assay. B) the Genomic DNA Ladder vial was shaken by manually inverting the tube 30 times. Degradation of the top fragment (48,500 bp) is clearly shown.

7

Degradation of the Genomic DNA Ladder as shown in Figure 11, can result in inaccurate sizing results. In instances where the degradation has resulted in failure of the software to assign the top ladder fragment, no DIN score will be presented for the lanes in this fi le. Where the ladder failure has resulted in a loss of data, a software alert will be presented in the sample table and above the gel lane.

Conclusion

The Agilent 2200 TapeStation system provides scalable throughput and automation, making it an ideal solution for quality control of biological samples in next-generation sequencing (NGS), microarray, and quantitative PCR workfl ows.

Following the good measurement practices described in this Technical

Overview ensures reliable and accurate

DNA quantifi cation and sizing.

References

1. Agilent 2200 TapeStation User Manual,

Agilent Technologies, publication number G2964-90003, 2013.

2. Agilent D1000 ScreenTape System

Quick Guide, Agilent Technologies, publication number G2964-90032,

2013.

3. Agilent High Sensitivity D1000

ScreenTape System Quick Guide,

Agilent Technologies, publication number G2964-90131, 2013.

4. Agilent Genomic DNA ScreenTape

System Quick Guide, Agilent

Technologies, publication number

G2964-90040, 2012.

5. Agilent D5000 ScreenTape System

Quick Guide, Agilent Technologies, publication number G2964-90050,

2015.

6. Agilent High Sensitivity D5000

ScreenTape System Quick Guide,

Agilent Technologies, publication number G2964-90150, 2015.

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., 2015

Published in the USA, September 1, 2015

5991-5153EN