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Technical OverviewPerformance Equivalence of theD1000 ScreenTape Assays on theAgilent TapeStation SystemsIntroductionAgilent TapeStation systems are well-established automated electrophoresissystems 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 withapplication-specific ScreenTape sample-processing consumable devices.DNA and RNA samples can be analyzed with minimal handling effort and fullyscalable throughput from 1 to 96 samples. The entire Agilent ScreenTapeportfolio for TapeStation systems is a vital tool for sample quality control (QC) atdifferent checkpoints in next-generation sequencing (NGS) workflows.To provide users with continued improvements and benefits to the TapeStationplatform, Agilent has recently introduced a technology update to the Agilent4200 TapeStation instrument. For example, all instruments manufactured fromspring 2021 are equipped with a modified ScreenTape nest, enabling users toeasily exchange the electrode cartridge during the yearly, mandatory, preventivemaintenance service. Thus, the modified ScreenTape nest secures an optimizedcost of ownership. In addition, a modified instrument optical system allowsfor a better match with the application-specific fluorescent dyes. All existingScreenTape applications as well as plastic consumables are fully compatiblewith the new 4200 TapeStation system.This technical overview highlights the performance of the Agilent D1000ScreenTape assay and the Agilent High Sensitivity D1000 ScreenTape assayon the new 4200 TapeStation system. Analytical assay specifications likesensitivity, sizing, quantification, and molarity delivered by the new instrumentwere evaluated and compared with the results obtained on the legacy 4200 and4150 TapeStation systems to demonstrate data equivalency between all threemodels.

Analytical specificationsSizing, quantification, and molaritywere compared between threeTapeStation models using the providedcorresponding ScreenTape ladders assamples, a 300 bp DNA fragment, andsheared human genomic DNA (gDNA).Table 1 summarizes the analyticalspecifications of the D1000 and HighSensitivity D1000 ScreenTape assays.ExperimentalMaterialsThe 4150 TapeStation (p/n G2992AA),legacy 4200 TapeStation (p/n G2991AA),and new 4200 TapeStation (p/nG2991BA) systems with the AgilentD1000 ScreenTape (p/n 5067-5582),D1000 Reagents (p/n 5067-5583), AgilentHigh Sensitivity D1000 ScreenTape (p/n5067-5584), and High Sensitivity D1000Reagents (p/n 5067-5585) were obtainedfrom Agilent Technologies Inc. NoLimits300 bp DNA fragments (p/n SM1621),the Qubit 2.0 Fluorometer, and Qubit1X dsDNA HS (High Sensitivity) AssayKit (p/n Q33231) were purchased fromThermo Fisher Scientific Inc. Humangenomic DNA (p/n G304A) was acquiredfrom Promega. The M220 Focusedultrasonicator and the microTUBEs wereobtained from Covaris Inc.Sample preparationDNA fragments were prepared bydiluting the commercially availableDNA from Promega in 10 mM TrisHCl (pH 8.0) to achieve a desirednumber of concentrations within thequantitative ranges of both D1000ScreenTape assays (Table 1). HumangDNA provided by the vendor was2Table 1. Comparison of analytical specifications of the Agilent D1000 ScreenTape assay and the AgilentHigh Sensitivity D1000 ScreenTape assay.Analytical SpecificationsSizing RangeTypical ResolutionSensitivity1Agilent D1000ScreenTape AssayAgilent High Sensitivity D1000ScreenTape Assay35 to 1,000 bp35 to 1,000 bp35 to 300 bp: 15%300 to 1,000 bp: 10%35 to 300 bp: 15%300 to 1,000 bp: 10%0.1 ng/µL5 pg/µLSizing Precision25% CV5% CVSizing Accuracy2,3 10% 10%Quantitative Precision0.1 to 1 ng/µL: 15% CV1 to 50 ng/µL: 10% CV15% CVQuantitative Accuracy2 20% 20%0.1 to 50 ng/µL10 to 1,000 pg/µLQuantitative Range¹Signal-to-noise 3 (single peak)²Measured using one ladder per ScreenTape device3Sizing accuracy for analysis with electronic ladder: 20%utilized to generate DNA smears. ThegDNA was sheared in the microTUBEson the Covaris instrument according tothe manufacturer’s recommendations.A shearing time of 110 seconds wasselected to achieve a target peak sizeof approximately 300 bp. The finalsmear sample was diluted in 10 mMTris-HCl (pH 8.0) to prepare differentconcentrations satisfying the respectivespecifications1. Nominal concentrationsof all analyzed samples were determinedon the Qubit Fluorometer with Qubit 1XdsDNA HS Assay Kit. The respectiveDNA ScreenTape ladders were usedas samples without any additionaltreatment.DNA analysisThe D1000 and High Sensitivity D1000ScreenTape assays were utilized forsample analysis on three new 4200TapeStation instruments, and on singlelegacy 4200 and 4150 TapeStationsystems, respectively. Samplepreparation for both assays wereperformed according to the Agilent quickguide instructions2,3. The DNA sampleswere analyzed in replicates of nine onall TapeStation systems using AgilentTapeStation software 4.1.

Results and discussionSensitivity750 pg/µL250 pg/µL100010 pg/µL800600400Upper markerLower marker200Size[bp]1500100070050040030020010050025An enlarged section shows the overlayof the technical replicates (n 9) at aconcentration of 5 pg/µL, correspondingto the specified limit of detection(Table 1). The respective fragment peakof 300 bp was clearly detected for all9 replicates with signal-to-noise ratiogreater than 3. Thereby, the sensitivity ofthe High Sensitivity D1000 ScreenTapeassay using a commercially availablefragment was confirmed on the new4200 TapeStation system. Likewise, thesensitivity of 0.1 ng/µL was verified forthe D1000 ScreenTape assay (data notshown).1,000 pg/µL5 pg/µL500 pg/µLSample Intensity [Normalized FU]A dilution series of a 300 bp DNAfragment with six concentrations from 5to 1,000 pg/µL was analyzed on a new4200 TapeStation system using the HighSensitivity D1000 ScreenTape assay.The electropherogram overlay of all usedconcentrations demonstrated single anddistinct peaks (Figure 1).1200Figure 1. A 300 bp DNA fragment dilution series (5 to 1,000 pg/µL) was analyzed using the Agilent HighSensitivity D1000 ScreenTape assay on the new Agilent 4200 TapeStation system. The enlarged imageshows the overlay of individual electropherograms at the specified limit of detection of 5 pg/µL (n 9).3

Absolute sizes were determined on threenew 4200 TapeStation instruments andcompared with the results obtainedon both the legacy 4200 and 4150TapeStation systems for all fragmentsconstituting the correspondingladders. The sizing performance of allinstruments is presented as an individualbar chart for each assay with nominalladder fragment sizes supplied by Agilenton the X-axes (Figure 2).Agilent D1000 ScreenTape assay1,000800Determined size (bp)The analytical specifications of theD1000 and High Sensitivity D1000ScreenTape assays are summarized inTable 1. Differing in quantitative range,both D1000 assays allow for accurateseparation of DNA fragments andsmears ranging from 35 to 1,000 bp inlength. Previously, sizing specificationsof the D1000 DNA assays were evaluatedon the legacy 4200 TapeStationsystem using two commerciallyavailable ladders4. In this study,sizing performance on the new 4200TapeStation instrument was evaluatedusing the D1000 and High SensitivityD1000 ScreenTape assay ladders assamples, as described for the 4150TapeStation system5, and the results ofall TapeStation models were al size (bp)B4150 TapeStationLegacy 4200 TapeStationNew 4200 TapeStation (1)New 4200 TapeStation (2)New 4200 TapeStation (3)Agilent High Sensitivity D1000 ScreenTape assay1,000Determined size minal size (bp)4150 TapeStationLegacy 4200 TapeStationNew 4200 TapeStation (1)New 4200 TapeStation (2)New 4200 TapeStation (3)Figure 2. Sizing results for eight DNA fragments of the corresponding Agilent ScreenTape assay ladderanalyzed on three new Agilent 4200 TapeStation instruments and on both the legacy Agilent 4200TapeStation instrument and the Agilent 4150 TapeStation instrument (n 9) compared to nominal sizes.(A) Agilent D1000 ScreenTape assay and (B) Agilent High Sensitivity D1000 ScreenTape assay.4

]1500100070050040030020010050025The average size of a sample is a criticalparameter of an NGS library to beprepared for multiplex sequencing, as itis required for molarity calculation. Theaverage size of a library can be easilydetermined with the region analysisfunction of the TapeStation analysissoftware. In comparison to peak sizedetermination, which is well suited for asymmetrical size distribution, the regionanalysis is more accurate for shearedDNA with a tailing on either side. Aset region, flanking the entire smear,provides an average smear size thatexcludes a potential bias associatedwith uneven size distribution. Figure 3illustrates how the region functionality ofthe TapeStation system can be applied toa sheared DNA sample. The average sizeof a smear is automatically reported bythe TapeStation analysis software in theregion table.1000Sample Intensity [Normalized FU]Sizing accuracy and precision for theD1000 and the High Sensitivity D1000ScreenTape assays were evaluated onthe new 4200 TapeStation instrumentsin direct comparison with valuesobtained on the legacy 4200 andthe 4150 TapeStation instruments.Sizing accuracy was 2.6% or lessfor both ScreenTape assays on allTapeStation instruments, well withinassay specifications ( 10%). Sizingprecision did not exceed a coefficient ofvariation (%CV) of 1.1% for the D1000,and 2.1% for the High Sensitivity D1000ScreenTape assays on all three differentTapeStation models, which is within thespecified sizing precision of 5% for bothassays.Figure 3. An example electropherogram of a sheared DNA sample analyzed on the new Agilent 4200TapeStation system with the Agilent High Sensitivity D1000 ScreenTape assay using Agilent TapeStationanalysis software region function. The region from 45 to 1,000 bp was established to flank the entiresmear.5

Figure 4 shows the comparison of sizingresults of the D1000 and High SensitivityD1000 ScreenTape assays delivered bythe 4150, legacy 4200, and three new4200 TapeStation systems, respectively.The results of all TapeStation modelswere comparable throughout thenominal concentrations and highlyconsistent between the three new 4200TapeStation instruments. The D1000and High Sensitivity D1000 ScreenTapeassays demonstrated reproducible sizingwith a maximum %CV of 2.6% and 3.7%,respectively. Both DNA ScreenTapeassays met the specified analyticalspecifications of 5%.Accurate and precise sizing performanceof the D1000 and the High SensitivityD1000 ScreenTape assays was fullyvalidated with multiple sample typeson the new 4200 TapeStation system.The sizing results obtained on the newinstrument were equivalent to the resultsdelivered by the other two TapeStationmodels.AAgilent D1000 ScreenTape assay400350Average size (bp)300250200150100500550100Nominal concentration (ng/µL)B4150 TapeStationLegacy 4200 TapeStationNew 4200 TapeStation (1)New 4200 TapeStation (2)New 4200 TapeStation (3)Agilent High Sensitivity D1000 ScreenTape assay400350Average size (bp)Sheared gDNA samples of differentconcentrations were prepared tobe within the previously specifiedquantitative range for smears1. Thesamples were separated with boththe D1000 and the High SensitivityD1000 ScreenTape assays on all threeTapeStation models and demonstratedexcellent sizing performance withrespect to smear analysis. An identicalsmear analysis region ranging from 45 to1,000 bp was set for both assays withinthe TapeStation analysis software.3002502001501005005007,50015,000Nominal concentration (pg/µL)4150 TapeStationLegacy 4200 TapeStationNew 4200 TapeStation (1)New 4200 TapeStation (2)New 4200 TapeStation (3)Figure 4. Comparative analysis of average size for sheared DNA throughout the respective dilution series.(A) Agilent D1000 ScreenTape assay. (B) Agilent High Sensitivity D1000 ScreenTape assay.6