Comparison of DNA Assays Using the 4200 TapeStation System and 2100 Bioanalyzer System

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1 Technical Overview Comparison of DN ssays Using the TapeStation System and 1 ioanalyzer System Introduction The gilent 1 ioanalyzer system is a well established system for DN quality control in multiple workflows. Specifically in NGS workflows, the sample throughput has dramatically increased, creating the need for high-throughput systems for DN sample quality control. The gilent TapeStation system has scalable throughput from 1 to 96 samples and walk away operation, which are essential features for high-throughput analysis. The TapeStation system fully automates sample processing for DN electrophoresis, including sample loading, separation, and imaging. oth platforms offer several DN assays appropriate for a wide size and concentration range for the analysis of PCR products, fragmented DN, and DN libraries 1. This Technical Overview compares the performance of the gilent D1 ScreenTape assay and gilent High Sensitivity D1 ScreenTape assay (HS D1 ScreenTape assay) analyzed on the TapeStation system directly with the gilent DN 1 assay and gilent High Sensitivity DN assay (HS DN assay) of the 1 ioanalyzer system. nalytical specifications were compared by evaluating accuracy and precision of quantification and sizing with a suitable sample set of DN fragments and sheared DN.

2 nalytical Specifications Table 1 summarizes the analytical specifications of the D1 and HS D1 ScreenTape assays for the TapeStation system and the specifications of the DN 1 and HS DN assays for the 1 ioanalyzer system. The analytical specifications of the ScreenTape assays were previously systematically validated for sensitivity, sizing, quantification, and molarity. Experimental Technical Details The TapeStation system (p/n G991) with D1 ScreenTape (p/n 67-8) and reagents (p/n 67-8), High Sensitivity D1 ScreenTape (p/n 67-8) and reagents (p/n 67-8), as well as the 1 ioanalyzer system (p/n G9C) using the DN 1 Kit (67-1) and High Sensitivity DN kit (p/n 67-66) were obtained from gilent Technologies (Waldbronn, Germany). NoLimits DN fragments and lambda DN were purchased from Thermo Fisher Scientific Inc. (Waltham, M, US). Lambda DN was sheared with the M Focused ultrasonicator from Covaris, Inc. (Woburn, M, US) to produce DN smear samples with a size distribution between and 1, bp. Unless stated, the manufacturer s protocols and guidelines were followed. DN samples were diluted with water to match the specified quantitative range of the DN assays (Table 1). Table 1. nalytical specifications of the D1 and HS D1 ScreenTape assays ( TapeStation system) and the DN 1 and HS DN assays (1 ioanalyzer system). nalytical specifications D1 ScreenTape ssay gilent TapeStation System High Sensitivity D1 ScreenTape ssay gilent 1 ioanalyzer System DN 1 ssay High Sensitivity DN ssay Sizing range 1, bp 1, bp 1, bp 7, bp Sizing accuracy* ±1 %** ±1 %** ±1 % ±1 % Sizing precision* % CV % CV % CV % CV Quantitative range (DN fragments) Quantitative range (DN smears).1 ng/µl 1 1, pg/µl. ng/µl* pg/µl* 1 ng/µl. 1 ng/µl ng/µl Quantitative accuracy ± %*** ± %*** ± %* ± %* Quantitative precision.1 1 ng/µl: 1 % CV 1 ng/µl: 1 % CV 1 % CV bp: 1 % CV* 1, bp: % CV*, bp: ± 1 %*, 7, bp: ± 1 %* * Determined by analyzing the respective ladder as sample. ** ccuracy of software ladder ± %. *** Measured against the gilent TapeStation system.

3 Results and Discussion Sizing The D1 and HS D1 ScreenTape assays as well as the DN 1 assay allow the analysis of DN samples with a maximal size of 1, bp. Conversely, the HS DN assay of the 1 ioanalyzer system provides a wider sizing range, from 7, bp. The sizing performance for the ScreenTape assays shown in Table 1 were previously evaluated with two commercially available DN ladders. To compare the sizing of the ScreenTape assays for the TapeStation system directly with the 1 ioanalyzer assays, three DN fragments (,, and 1, bp) were diluted to match the quantitative range of each assay, then analyzed. s recommended by the D1 and HS D1 ScreenTape assay protocol, a ladder was run on each ScreenTape. The sizing results were plotted against the nominal sizes supplied by the manufacturer (Figure 1). The specified sizing accuracy (±1 %) was met for each DN fragment with both electrophoresis platforms and all assays. The D1 and HS D1 ScreenTape assays showed sizing accuracy similar to the DN 1 and HS DN assays with the 1 ioanalyzer system. Figure shows sizing precision evaluated with six replicates per fragment on each assay. The determined sizing precision for the D1 ScreenTape assay ( TapeStation system) and the DN 1 assay (1 ioanalyzer system) for all fragments was below 1 % CV, which is within the specified range for both assays ( % CV). The sizing precision of the HS D1 ScreenTape assay for all fragments was below 1 % CV and below % for the HS DN assay, which met the specifications of each assay ( % CV). Determined size Figure 1. Sizing results for three DN fragments (n = 6) analyzed with the TapeStation and 1 ioanalyzer systems. ) Sizing results of the D1 ScreenTape assay compared with the DN 1 assay. ) Sizing results comparing the High Sensitivity D1 ScreenTape assay with the High Sensitivity DN assay. % CV 1, 1, D1 ScreenTape assay DN 1 assay 1, D1 ScreenTape assay DN 1 assay 1, Determined size The results for sizing obtained with the TapeStation system showed equivalent accuracy and precision compared to the 1 ioanalyzer system. % CV 1, 1, HS D1 ScreenTape assay HS DN assay 1, HS D1 ScreenTape assay HS DN assay 1, Figure. Sizing precision analyzed with three different size DN fragments (n = 6) with the TapeStation and 1 ioanalyzer systems. ) Sizing precision of the D1 ScreenTape assay compared with the DN 1 assay. ) Sizing precision of the High Sensitivity D1 ScreenTape assay in comparison to the High Sensitivity DN assay.

4 Quantification The quantification performance for sensitivity, precision, and accuracy was previously validated for the D1 and HS D1 ScreenTape assays, and showed excellent performance. The quantitative range for the D1 ScreenTape assay is specified from.1 to ng/μl. This range provides a slightly higher sensitivity to the corresponding DN 1 assay of the 1 ioanalyzer system for sample concentrations between. and ng/µl, as shown in Table 1. Quantification of DN fragments with the HS D1 ScreenTape assay ranges from 1 to 1, pg/µl, whereas the HS DN assay of the 1 ioanalyzer system is suitable for concentrations between and pg/µl (Table 1). Quantification performance was compared between the assays by analyzing three dilutions of a bp DN fragment to cover low, mid, and high concentrations within the range of the assays. The quantitative precision of all samples met the specification of the respective assay (data not shown). The TapeStation system results correlated well with the 1 ioanalyzer results, as shown in Figure. Molarity Sample molarity is often used to determine the load volume of NGS libraries for pooling before sequencing. The calculation of molarity is based on average size and concentration of library samples. Thus, for accurate molarity results and successful library sequencing, sizing and quantification must be accurate. Excellent assay performance for NGS library samples was shown previously for the D1 and HS D1 ScreenTape assays. It was also demonstrated that the assays are suitable for sample quality control in a high throughput sequencing environment,. y =.918x y = 1.17x 9.8 R² =.99 R² = Concentration DN 1 assay (ng/µl) Concentration D1 ScreenTape assay (ng/µl) Concentration High Sensitivity DN assay (pg/µl) The lower and upper size limits of the DN assays (Table 1) apply for maximum peak sizes of DN fragments. DN libraries must match the assay range with the whole sample size to fit between the lower and upper marker of an assay. Table 1 shows that the quantitative range for DN smears differs from the quantitative range for DN fragments. dilution series of sheared DN was analyzed with the D1 and HS D1 ScreenTape assays on the TapeStation system. ladder was run on each ScreenTape to ensure the most accurate sizing Concentration High Sensitivity D1 ScreenTape assay (pg/µl) Figure. Quantification of a bp fragment in three concentrations (n = 6) with the TapeStation system (on the X-axis) compared to the 1 ioanalyzer system (on the Y-axis). ) Concentrations measured with the D1 ScreenTape assay compared to concentrations obtained from the DN 1 assay. ) Comparison of concentrations analyzed with the HS D1 ScreenTape assay and with the HS DN assay.

5 The molarity data was directly compared to the data of the same samples analyzed with the DN 1 and HS DN assays of the 1 ioanalyzer system. The results showed excellent correlation for molarity analysis of sheared DN with the TapeStation system compared to the 1 ioanalyzer system (Figure ). Figures and 6 show that the electropherogram patterns of the ScreenTape assays for DN smears are not identical to the pattern obtained with the 1 ioanalyzer system. This effect occurs due to different separation processes during gel electrophoresis on the ioanalyzer chips and the ScreenTape devices. The position of the region on the X-axis depends on the size range of the assay. utomatic integration and data processing of the TapeStation nalysis software and the 1 Expert software generate highly comparable results for sizing, quantification, and molarity analysis of sheared DN samples measured with the TapeStation and the 1 ioanalyzer systems. Molarity DN 1 assay (nmol/l) y = 1.1x R² = Molarity D1 ScreenTape assay (nmol/l) Figure. Correlation of molarity data of sheared DN samples in three concentrations (n = 6). The results of the TapeStation system are plotted on the X-axis and the results obtained with the 1 ioanalyzer system on the Y-axis. The molarity was evaluated using region functionality. ) Sample molarities obtained with the D1 ScreenTape assay compared with molarity data from the DN 1 assay. ) Correlated molarity data of sheared DN samples analyzed with the HS D1 ScreenTape assay and the HS DN assay. Sample intensity (normalized FU),,,, 1, Lower 8 8 Upper 1 Molarity High Sensitivity DN assay (pmol/l) FU y = 1.x R² = Molarity High Sensitivity D1 ScreenTape assay (pmol/l) 1 Sheared 1 7 1, 1, , Figure. Electropherogram pattern of sheared DN analyzed with the TapeStation and 1 ioanalyzer systems. ) Electropherogram of sheared DN separated with the D1 ScreenTape assay. ) Electropherogram of the same sample obtained with the DN 1 assay. Lower G1:G Upper FU G Sample intensity (normalized FU) , 1, 1 6, 1,8 Figure 6. Electropherogram patterns of sheared DN analyzed with the TapeStation and 1 ioanalyzer systems. ) Example electropherogram of a sheared DN sample analyzed with the HS D1 ScreenTape assay ) The same sample analyzed with the HS DN assay.

6 Conclusion This Technical Overview shows that sizing, quantification, and molarity data of DN ScreenTape assays for the gilent TapeStation system highly correlate with data of equivalent assays on the gilent 1 ioanalyzer system. oth systems show similar performance for sizing and quantification of DN fragments and smears. Electropherogram patterns of sheared DN analyzed with ScreenTape assays differ slightly from patterns obtained with corresponding assays of the 1 ioanalyzer system. The results for molarity, quantification, and sizing correlate highly between equivalent assays on both platforms for DN smears. For the most accurate sizing and molarity results, it is recommended to run a ladder on each ScreenTape. References 1. gilent ioanalyzer and TapeStation Systems - Systems, Consumables and Supplies. gilent Technologies, publication number EN, 1.. Performance of the gilent D1 and the gilent High Sensitivity D1 ScreenTape ssay for the gilent TapeStation System. gilent Technologies Technical Overview, publication number EN, 16.. Viering, E.; et al. Evaluating the gilent TapeStation System for High Throughput Sequencing Quality Control. gilent Technologies pplication Note, publication number EN, 16.. Petersen, J.; et al. Use of the gilent TapeStation System for Sample Quality Control in the Whole Exome Sequencing Workflow at the German Cancer Research Center (DKFZ). gilent Technologies pplication Note, publication number EN, For Research Use Only. Not for use in diagnostic procedures. This information is subject to change without notice. gilent Technologies, Inc. 18 Printed in the US, May 1, EN