Performance of the Agilent D1000 and the Agilent High Sensitivity D1000 ScreenTape Assay for the Agilent 4200 TapeStation System

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1 Performance of the gilent D and the gilent High Sensitivity D ScreenTape ssay for the gilent 42 TapeStation System Technical Overview Introduction The gilent 42 TapeStation system is an automated system for fast and reliable DN and RN electrophoresis. In comparison to the gilent 22 TapeStation system, it offers fully scalable throughput from to 96 samples, and increased walk away time, which are essential features for high throughput customers. The gilent D ScreenTape assays were developed to separate and analyze DN molecules ranging from 3 to, bp and provide size and concentration values. The 42 TapeStation system, together with the D ScreenTape assays, is ideally suited for the analysis of PCR products, fragmented DN, and DN libraries. This Technical Overview focuses on the performance of both D ScreenTape assays in evaluating the accuracy and precision of quantification and sizing, as well as the sensitivity of these assays. In addition, the data were compared to the 22 TapeStation system to demonstrate the full compatibility of both systems.

2 Table summarizes the analytical specifications of the D and the gilent High Sensitivity D ScreenTape assays. Experimental Material Two commercially available DN ladders (source undisclosed) and NoLimits DN fragments, the Qubit. Fluorometer, and NanoDrop were purchased from Thermo Fisher Scientific Inc. (Waltham, M, US). Mouse genomic DN was purchased from Promega, and the M22 Focused-ultrasonicator from Covaris, Inc. (Woburn, M, US). The gilent 42 TapeStation (p/n G299) and gilent 22 TapeStation (p/n G2964 or G296) systems with gilent D ScreenTape (p/n 67-82), gilent D ScreenTape Reagents (p/n 67-83), gilent High Sensitivity D ScreenTape (p/n 67-84), and gilent High Sensitivity D ScreenTape Reagents (p/n 67-8) were obtained from gilent Technologies (Waldbronn, Germany). Sample preparation The DN fragments and ladders were diluted with water so that the concentration fit into the specified quantitative range of both DN assays (Table ). Mouse gdn was sheared with an ultrasonicator to prepare the DN smear sample. DN analysis DN analysis was performed using the D or the High Sensitivity D ScreenTape assay in combination with the 22 and 42 TapeStation systems, according to the manufacturer s instructions -4. Table. nalytical specifications of the gilent D and the gilent High Sensitivity D ScreenTape assays for the gilent 42 TapeStation system. nalytical specifications gilent D ScreenTape ssay Sizing range 3, bp 3, bp Typical resolution 3 3 bp: % 3, bp: % gilent High Sensitivity D ScreenTape ssay 3 3 bp: % 3, bp: % Sensitivity. ng/μl pg/μl Sizing precision 2 % CV % CV Sizing accuracy 2,3 ± % ± % Quantitative precision. ng/μl: % CV % CV ng/μl: % CV Quantitative accuracy 4 ± 2 % ± 2 % Quantitative range. ng/μl, pg/μl Maximum sample buffer strength 2 mm KCl, 6 mm Phosphate buffer, 6 mm Guanidine-HCl, 24 mm NaCl, 6 mm cetate 7 mm KCl, 2 mm Phosphate buffer, 2 mm Guanidine-HCl, 8 mm NaCl, 2 mm cetate Signal-to-noise (S/N) > 3 (single peak) 2 Measured using one ladder per ScreenTape device 3 Sizing accuracy for analysis with electronic ladder: ± 2 % 4 Measured against the gilent 22 TapeStation system 2

3 Results and Discussion Sensitivity dilution series of a single 6 bp DN fragment from ng/µl to pg/μl was analyzed with the 42 TapeStation system using the High Sensitivity D ScreenTape assay. Figure shows the electropherogram overlay for this dilution series, where the signal peaks are clearly visible for all tested concentrations. The enlarged image shows the electropherogram overlay of the repeated analysis of pg/μl DN (n = 8). The peaks are clearly visible above the background signal, demonstrating the specified sensitivity of pg/µl for the gilent High Sensitivity D ScreenTape assay (Table ). The lower limit of detection for the D ScreenTape assay is specified to be. ng/µl for a single fragment at a signal-to-noise (S/N) ratio of larger than 3 (data not shown). DN Sizing To determine the sizing accuracy of both D ScreenTape assays, two commercially available DN ladders were analyzed with the 42 TapeStation system and the 22 TapeStation system. The obtained sizes were plotted against the nominal sizes as supplied by the manufacturers. Ten different DN fragments ranging from 8 to, bp, depending on the DN ladder, were analyzed. For all tested DN fragments, the determined sizing accuracy with the 42 TapeStation system and the 22 TapeStation system was below % for both the standard and the high sensitivity assays (Figure 2). Sample intensity (FU),6,4,2, Marker 2 pg/µl Marker, pg/µl 6 pg/µl 3 pg/µl pg/µl Figure. 6 bp DN fragment dilution series from ng/µl to pg/μl was analyzed with the gilent High Sensitivity D ScreenTape assay and the gilent 42 TapeStation system. The electropherogram overlay is shown. The enlarged image shows an overlay of eight individual electropherograms at a DN concentration of pg/μl. % Deviation from nominal size % Deviation from nominal size gilent D ScreenTape ssay 7,, Size [bp] gilent 42 TapeStation system (n = 78) gilent 22 TapeStation system (n = ) Nominal size (bp) gilent High Sensitivity D ScreenTape ssay gilent 42 TapeStation system (n = 77) gilent 22 TapeStation system (n = 2) , Nominal size (bp) Figure 2. The sizing accuracy of the DN analysis with the gilent 42 TapeStation and gilent 22 TapeStation systems was determined using two commercial DN ladders. The % deviation from the nominal sizes as supplied by the manufacturer is shown. 3

4 The sizing precision of both DN assays with the 42 TapeStation system, in comparison to the 22 TapeStation system, were evaluated and shown in Figure 3. The determined sizing precision for the DN analysis with standard sensitivity as well as high sensitivity assays was below % CV for both TapeStation systems (Figure 3, Table ). The % CV sizing precision specified for the D and the High Sensitivity D ScreenTape assay was determined using one DN ladder per ScreenTape device. The slightly higher %CV values for the 42 TapeStation system might be caused by using multiple tapes for this test compared to only one tape with the 22 TapeStation system. % CV gilent D ScreenTape ssay gilent 42 TapeStation system (n = 78) gilent 22 TapeStation system (n = ) Nominal size (bp) gilent High Sensitivity D ScreenTape ssay gilent 42 TapeStation system (n = 77) gilent 22 TapeStation system (n = 2) This excellent sizing precision is not only obtained for a commercial DN ladder, but can also be achieved when analyzing a more complex DN sample. To prove this, a DN sample with a distribution of multiple fragment sizes (smear), typical for samples within the Next Generation Sequencing (NGS) workflows, was analyzed with the D and the High Sensitivity D ScreenTape assay and the 42 TapeStation system at different concentrations across the specified concentration range of the two assays. The obtained sizing precision for the DN smear sample was below 3 % CV for all tested concentrations (data not shown). % CV , Nominal size (bp) Figure 3. The sizing precision of the standard and high sensitivity DN analysis with the gilent 42 TapeStation system and the gilent 22 TapeStation system was determined using commercial DN ladders. 4

5 DN Quantification In addition to sizing, the D and High Sensitivity D ScreenTape assays also provide quantification data for individual DN fragments and smears within a sample. To demonstrate the quantification accuracy and precision of the 42 TapeStation system and its correlation with the 22 TapeStation system, a dilution series of a 6 bp DN fragment was quantified with both systems. For the D ScreenTape assay, the concentration ranged from. to ng/µl, and for the High Sensitivity ScreenTape assay from to, pg/µl. The DN concentrations determined with both TapeStation systems were plotted against each other using a logarithmic scale (Figure 4). Figure 4 shows excellent correlation, with R 2 values of 99.9 % for DN quantification of a single 6 bp DN fragment between the 42 TapeStation system and the 22 TapeStation system for both the D and the High Sensitivity D ScreenTape assays. The quantification accuracy for the 42 TapeStation system, as indicated by the % deviation from the concentrations obtained with the 22 TapeStation system, was below 4 % for the D ScreenTape assay and below 6 % for the High Sensitivity D ScreenTape assay. The specified quantification accuracy for both D assays is ± 2 % measured against the 22 TapeStation system (Table ). gilent 22 TapeStation system concentration (pg/µl) gilent 22 TapeStation system concentration (pg/µl). gilent D ScreenTape assay gilent High Sensitivity D ScreenTape assay y =.973x R 2 =... gilent 42 TapeStation system concentration (pg/µl), y =.962x R 2 =.9998, gilent 42 TapeStation system concentration (pg/µl) Figure 4. The concentrations of a 6 bp DN fragment dilution series from the gilent D and gilent High Sensitivity D ScreenTape assays on the gilent 42 TapeStation system (n = 9 to 2) and the gilent 22 TapeStation system (n = to 6) system were plotted against each other on a logarithmic scale. Dilutions of a 3 bp DN fragment were used to verify the DN quantification precision across different concentrations on the 42 TapeStation and 22 TapeStation systems.

6 The determined quantification precision for the DN analysis with standard sensitivity was below % CV for concentrations from to ng/µl, and below % CV at. ng/µl (Figure ). The quantification precision for the high sensitivity DN analysis was below % CV for all tested concentrations (Figure 6). Therefore, the calculated quantification precision for the D ScreenTape and the High Sensitivity D ScreenTape assays on the 42 TapeStation system were well within specifications (Table ). % CV D ScreenTape assay. 2 Nominal sample concentration (ng/µl) High Sensitivity D ScreenTape assay gilent 42 TapeStation system (n = 6 63) gilent 22 TapeStation system (n = 4 2) gilent 42 TapeStation system (n = 73 8) gilent 22 TapeStation system (n = 4 8) % CV, Nominal sample concentration (ng/µl) Figure. Quantification precision for a 3 bp DN fragment dilution series from the gilent D ScreenTape and gilent High Sensitivity D ScreenTape assays on the gilent 42 and gilent 22 TapeStation systems. 6

7 Molarity The molarity of DN samples is important in determining the input amount of DN for subsequent sequencing reactions for NGS protocols. DN smear sample was used to determine the correlation of the molarity values obtained with the 42 TapeStation and 22 TapeStation systems for the D and the High Sensitivity D ScreenTape assays (Figure 6). Figure 6 shows excellent correlation, with R 2 values of 99.9 % for the molarity of a DN smear sample analyzed on the 42 TapeStation and 22 TapeStation systems with both the D and the High Sensitivity D ScreenTape assays. gilent 22 TapeStation system molarity (nmol/µl) gilent 22 TapeStation system molarity (nmol/µl), gilent D ScreenTape assay y =.972x R 2 = , gilent 42 TapeStation system molarity (nmol/l) 4 2 gilent D High SensitivityScreenTape assay y =.64x R 2 =.9998,,, 2, gilent 42 TapeStation system molarity (nmol/l) Figure 6. Molarity correlation of a DN smear dilution series determined with the gilent D and gilent High Sensitivity D ScreenTape assays on the gilent 42 TapeStation and gilent 22 TapeStation systems (n = to 2). 7

8 Conclusion This Technical Overview shows that the gilent D and the gilent High Sensitivity D ScreenTape assays for the gilent 42 TapeStation system provide efficient and reliable DN analysis of DN fragments, including highly accurate and reproducible sizing and quantification. Furthermore, it demonstrates that the D ScreenTape assays can also be applied to determine the DN concentration and molarity of DN smear samples. The data obtained with the 42 TapeStation system exhibits equivalency with the data generated from the gilent 22 TapeStation system. In contrast to the 22 TapeStation system, the 42 platform maximizes efficiency by offering electrophoresis of up to 96 samples in an unattended manner. References. D ScreenTape System Quick Guide, gilent Technologies, publication number G Rev. D, High Sensitivity D ScreenTape System Quick Guide, gilent Technologies, publication number G Rev. D, D ScreenTape ssay Quick Guide for 42 TapeStation System, gilent Technologies, publication number G299-93, High Sensitivity D ScreenTape ssay Quick Guide for 42 TapeStation System, gilent Technologies, publication number G299-93, 2. tapestation For Research Use Only. Not for use in diagnostic procedures. This information is subject to change without notice. gilent Technologies, Inc., 26 Published in the US, June, EN