Agilent D1000 User Manual

Technical Overview

Performance Equivalence of the D1000 ScreenTape Assays on the Agilent TapeStation Systems

Introduction

Agilent TapeStation systems are well-established automated electrophoresis systems for fast and reliable analysis of nucleic acids for multiple applications. The platform integrates a benchtop electrophoresis-based instrument, data-processing TapeStation software, and reagents, used in concert with application-specific ScreenTape sample-processing consumable devices. DNA and RNA samples can be analyzed with minimal handling effort and fully scalable throughput from 1 to 96 samples. The entire Agilent ScreenTape portfolio for TapeStation systems is a vital tool for sample quality control (QC) at different checkpoints in next-generation sequencing (NGS) workflows.

To provide users with continued improvements and benefits to the TapeStation platform, Agilent has recently introduced a technology update to the Agilent 4200 TapeStation instrument. For example, all instruments manufactured from spring 2021 are equipped with a modified ScreenTape nest, enabling users to easily exchange the electrode cartridge during the yearly, mandatory, preventive maintenance service. Thus, the modified ScreenTape nest secures an optimized cost of ownership. In addition, a modified instrument optical system allows for a better match with the application-specific fluorescent dyes. All existing ScreenTape applications as well as plastic consumables are fully compatible with the new 4200 TapeStation system.

This technical overview highlights the performance of the Agilent D1000 ScreenTape assay and the Agilent High Sensitivity D1000 ScreenTape assay on the new 4200 TapeStation system. Analytical assay specifications like sensitivity, sizing, quantification, and molarity delivered by the new instrument were evaluated and compared with the results obtained on the legacy 4200 and 4150 TapeStation systems to demonstrate data equivalency between all three models.

Analytical specifications

Sizing, quantification, and molarity were compared between three TapeStation models using the provided corresponding ScreenTape ladders as samples, a 300 bp DNA fragment, and sheared human genomic DNA (gDNA). Table 1 summarizes the analytical specifications of the D1000 and High Sensitivity D1000 ScreenTape assays.

Experimental

Materials

The 4150 TapeStation (p/n G2992AA), legacy 4200 TapeStation (p/n G2991AA), and new 4200 TapeStation (p/n G2991BA) systems with the Agilent D1000 ScreenTape (p/n 5067-5582), D1000 Reagents (p/n 5067-5583), Agilent High Sensitivity D1000 ScreenTape (p/n 5067-5584), and High Sensitivity D1000 Reagents (p/n 5067-5585) were obtained from Agilent Technologies Inc. NoLimits 300 bp DNA fragments (p/n SM1621), the Qubit 2.0 Fluorometer, and Qubit 1X dsDNA HS (High Sensitivity) Assay Kit (p/n Q33231) were purchased from Thermo Fisher Scientific Inc. Human genomic DNA (p/n G304A) was acquired from Promega. The M220 Focusedultrasonicator and the microTUBEs were obtained from Covaris Inc.

Sample preparation

DNA fragments were prepared by diluting the commercially available DNA from Promega in 10 mM TrisHCl (pH = 8.0) to achieve a desired number of concentrations within the quantitative ranges of both D1000 ScreenTape assays (Table 1). Human gDNA provided by the vendor was

Table 1. Comparison of analytical specifications of the Agilent D1000 ScreenTape assay and the Agilent High Sensitivity D1000 ScreenTape assay.

Analytical Specifications Agilent D1000

Sizing Range 35 to 1,000 bp 35 to 1,000 bp

Typical Resolution

Sensitivity

Sizing Precision

Sizing Accuracy

Quantitative Precision

Quantitative Accuracy

Quantitative Range 0.1 to 50 ng/µL 10 to 1,000 pg/µL

¹Signal-to-noise >3 (single peak) ²Measured using one ladder per ScreenTape device

2,3

Sizing accuracy for analysis with electronic ladder: ±20%

utilized to generate DNA smears. The gDNA was sheared in the microTUBEs on the Covaris instrument according to the manufacturer’s recommendations. A shearing time of 110 seconds was selected to achieve a target peak size of approximately 300 bp. The final smear sample was diluted in 10 mM Tris-HCl (pH = 8.0) to prepare different concentrations satisfying the respective specifications1. Nominal concentrations of all analyzed samples were determined on the Qubit Fluorometer with Qubit 1X

ScreenTape Assay

35 to 300 bp: 15%

300 to 1,000 bp: 10%

0.1 ng/µL 5 pg/µL

5% CV 5% CV

±10% ±10%

0.1 to 1 ng/µL: 15% CV 1 to 50 ng/µL: 10% CV

±20% ±20%

DNA analysis

The D1000 and High Sensitivity D1000 ScreenTape assays were utilized for sample analysis on three new 4200 TapeStation instruments, and on single legacy 4200 and 4150 TapeStation systems, respectively. Sample preparation for both assays were performed according to the Agilent quick guide instructions were analyzed in replicates of nine on all TapeStation systems using Agilent TapeStation software 4.1.

Agilent High Sensitivity D1000

ScreenTape Assay

35 to 300 bp: 15%

300 to 1,000 bp: 10%

15% CV

2,3

. The DNA samples

dsDNA HS Assay Kit. The respective DNA ScreenTape ladders were used as samples without any additional treatment.

Sample Intensity [Normalized FU]

[bp]

1,000 pg/µL

Results and discussion

Sensitivity

A dilution series of a 300 bp DNA fragment with six concentrations from 5 to 1,000 pg/µL was analyzed on a new 4200 TapeStation system using the High Sensitivity D1000 ScreenTape assay. The electropherogram overlay of all used concentrations demonstrated single and distinct peaks (Figure 1).

An enlarged section shows the overlay of the technical replicates (n = 9) at a concentration of 5 pg/µL, corresponding to the specified limit of detection (Table 1). The respective fragment peak of 300 bp was clearly detected for all 9 replicates with signal-to-noise ratio greater than 3. Thereby, the sensitivity of the High Sensitivity D1000 ScreenTape assay using a commercially available fragment was confirmed on the new 4200 TapeStation system. Likewise, the sensitivity of 0.1 ng/µL was verified for the D1000 ScreenTape assay (data not shown).

1200

1000

800

600

400

200

Figure 1. A 300 bp DNA fragment dilution series (5 to 1,000 pg/µL) was analyzed using the Agilent High Sensitivity D1000 ScreenTape assay on the new Agilent 4200 TapeStation system. The enlarged image shows the overlay of individual electropherograms at the specified limit of detection of 5 pg/µL (n = 9).

5 pg/µL

Lower marker

750 pg/µL

500 pg/µL

250 pg/µL

10 pg/µL

Upper marker

Siz

Sizing

Agilent D1000 ScreenTape assay

Determined size (bp)

The analytical specifications of the D1000 and High Sensitivity D1000 ScreenTape assays are summarized in Table 1. Differing in quantitative range, both D1000 assays allow for accurate separation of DNA fragments and smears ranging from 35 to 1,000 bp in length. Previously, sizing specifications of the D1000 DNA assays were evaluated on the legacy 4200 TapeStation system using two commercially available ladders4. In this study, sizing performance on the new 4200 TapeStation instrument was evaluated using the D1000 and High Sensitivity D1000 ScreenTape assay ladders as samples, as described for the 4150 TapeStation system5, and the results of all TapeStation models were compared.

Absolute sizes were determined on three new 4200 TapeStation instruments and compared with the results obtained on both the legacy 4200 and 4150 TapeStation systems for all fragments constituting the corresponding ladders. The sizing performance of all instruments is presented as an individual bar chart for each assay with nominal ladder fragment sizes supplied by Agilent on the X-axes (Figure 2).

1,000

800

600

400

Determined size (bp)

200

Agilent High Sensitivity D1000 ScreenTape assay

1,000

800

600

400

50 100 200 300 400 500 700 1,000

Nominal size (bp)

4150 TapeStation

New 4200 TapeStation (1)

Legacy 4200 TapeStation

New 4200 TapeStation (2) New 4200 TapeStation (3)

200