Automated KAPA ® Library Quantifcation Kit with the epMotion ® 5075t 1Eppendorf Application Technologies SA, Namur, Belgium Abstract epMotion sample dilution accuracy was assessed using DNA Standard 0 diluted 1:10,000, as well as two NGS libraries diluted 1:10,000; 1:100,000; and 1:1,000,000. Same stock of reagents and samples were used in both manual and automated processes. The qPCR plate layout is illustrated on Figure 1. Figure 1: qPCR plate layout. Figure 2: epMotion 5075t worktable for sample dilution and qPCR setup with the KAPA Library Quantifcation Kit for Illumina ® platforms. dental chair accessories,chair accessories parts dental,dental chair spare parts,dental chair parts Foshan Ja Suo Medical Device Co., LTD , https://www.fjoralinstrument.com
Quantification of NGS libraries prior to sequencing is an important basis for obtaining reliable results and optimizing sequencing performance. This application demonstrates the outstanding performance of the epMotion 5075t Liquid Workstation, with complete accuracy and excellent accuracy for complete qPCR analysis in accordance with the operational requirements of the KAPA Library Quantitation Kit.
Maud Brasseur1, Jennifer Pavlica2, Marsha McMakin2, Sandrine Hamels1
2Roche Sequencing & Life Science, Wilmington, USA
Next-Generation Sequencing (NGS) is a high-throughput method that enables highly parallelized sequencing of multiple libraries, prepared from both DNA and RNA samples. Bolstered by growing applications, such as the whole genome, whole exome, and gene expression analyses, NGS Is gaining importance and becoming a standard procedure in many laboratories.
The quantifcation of NGS libraries prior to sequencing is essential to obtain reliable results and optimal sequencing performance. This Application Note demonstrates the capability of the epMotion 5075t liquid handling system to automate with high accuracy and high precision a complete qPCR assay setup using the KAPA Library Quantifcation Kit including library dilution steps.
Introduction
Accurate and precise quantifcation of the number of amplifable molecules in a library is an essential step in sequencing workflows. It ensures consistent and optimal cluster densities, as well as equivalent representation of each library when sequencing in multiplex.
Quantification can result in decreased sequence quality metrics, inefcient flowcell use, and additional sequencing required for underrepresented samples – all of which translates to wasted time and money.
A quantitative PCR (qPCR) approach, such as the KAPA Library Quantifcation Kit, is the gold standard for NGS library quantifcation and is the only method that accurately measures the number of amplifable molecules that have the ability to cluster. Sensitivity and broad dynamic range for accurate quantifcation of very
Dilute and PCR-free libraries.
Performing an accurate qPCR assay manually in a high durability setting becomes difcult and time consuming, and the use of an automated liquid handling platform is strongly recommended to reduce the risk of human error and increase the reliability of results. The Eppendorf epMotion ® family of automated The epMotion 5075t and other models in the Eppendorf family pipette volumes ranging from 0.2 μL to 1 mL with efciency and accuracy.
The reliability of qPCR results is highly relevant to the accuracy and precision of liquid handling in both reaction setup and library dilution. Accuracy and precision are two differences that are often used interchangeably, but are actually differentiated. Accuracy refers to how close a measurement is to a true value, while precision refers to how close repeat measurements are to each other and is thus a measure of reproducibility. This Application Note presents experimental results aimed at verifying the accuracy and precision of the KAPA Library Quantifcation Kit processed on the epMotion 5075t compared to Manual handling.
Materials and Methods
Materials
q PCR – Accessories
> LightCycler® 480 Sealing Foil (Roche, cat # 04 729 757 001)
> LightCycler® 480 Instrument, 96-well (Roche, cat # 05 015 278 001)
> Centrifuge MiniSpin®, non-refrigerated, with Rotor F-45-12-11, 230 V/50 – 60 Hz (Eppendorf, cat # 5452000010)
> Centrifuge 5810 R (IVD), refrigerated, without rotor, keypad, 230 V/50 – 60 Hz (Eppendorf, cat # 5811000015) qPCR – Reagents
> KAPA Library Quantifcation Kit for Illumina® platforms (KAPA Biosystems, cat # 07960336001)
> KAPA Library Quantifcation Dilution Control Kit for Illumina® platforms (KAPA Biosystems, cat # 07960417001)
> UltraPureTM 1M Tris-HCl, pH 8.0 (Thermo Fisher®, cat # 15568025) > Water, Sterile, Nuclease-free, Biotechnology Grade (VWR, cat # E476)
Methods
KAPA Library Quantifcation Kit contains all reagents required for quantifcation of NGS libraries for Illumina® sequencing, including KAPA SYBR® FAST qPCR Master Mix (2X), a Primer PreMix (10X) containing two primers based on Illumina P5 and P7 oligo sequences (Primer 1 : 5'-AAT GAT ACG GCG ACC ACC GA-3' and Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3') and six ready-to-use DNA standards corresponding to a 10-fold dilution series. Standards consist of a linear, 452 bp dsDNA fragment flanked by qPCR primer binding sites. A KAPA Library Quantifcation Dilution Control, referred to asDNA Standard 0, is a 200 pM solution of the same linear, 452 bp dsDNA fragment. It is available for purchase Between and can be used to characterize impact of liquid handling on assay accuracy.
The epMotion was programmed to perform NGS library sample dilution and qPCR setup in one method. Reactions were set up according to Table 1 and thermocycled according to Table 2 using a LightCycler® 480 instrument (96 well). Template-free negative controls were included. 
Before starting the program, the epMotion surfaces and tools were cleaned using a DNA decontamination solution "DNA Erase" and treated with UV-light for 15 minutes. The work table of epMotion 5075t instrument is equipped as in Figure 2.
Automated qPCR Setup Accuracy and Precision
The accuracy and precision of the qPCR setup was evaluated based on standard curve analysis (Figure 3). Amplifca tion efciency is equivalent between automated (95%) and manual (94%) setup, and both values ​​are within the acceptable range of 90 The standard of the s. Points were less than 0.1, translating to a coefcient of varia tion less than 0.4%. This indicates that any quantifcation data obtained is accurate, assuming accurate and precise library dilution. 
Automated Sample Dilution Accuracy and Precision
The standard pM value of the automated Standard 0 reactions (216 pM) is similar to the average pM value of the manual reactions (222) pM) showing equivalency of the automated sample dilution process on the epMotion (Figure 4).
Additionally, the automated process resulted in an estimated Standard 0 concentration of 216 pM, which deviates from the expected value of 200 pM by less than 10%. The low standard deviation (CV of 1.4%) among the twelve replicates shows high precision and highlights The reliability of the automated sample dilution process. 
As was for a 10-fold dilution series, the Delta Cq values ​​between the different dilution Factors were within the acceptable range of 3.1 to 3.6. The Cq standard deviation of both NGS libraries does not exceed 0.2% showing high reproducibility (Figure 5). 
Conclusions
The automation of the KAPA Library Quantifcation Kit on the Eppendorf epMotion 5075t offers a solution for high throughput quantifcation, reducing hands-on time and human error with a total runtime of only 28 minutes for the here presented setup. The Eppendorf epMotion liquid han dling systems Have the advantage of having a user friendly interface, with flexible options for modifying the number of libraries, the number of qPCR replicates, and the dilution factor to desired experimental design with a maximum of 72 reactions per plate. Reliable, optimized and ready-to -use epMotion methods are available for small epMotion mod els such as the epMotion 5073 and larger sizes such as the epMotion 5075. The presence of a thermo-module on the worktable keeps the qPCR plate refrigerated during process ing, and detection of tip and consumable Priors before run start allows reliable processing without incident. 
Fully automated NGS library quantification based on epMotion 5075t and KAPA library quantification kits
Guide
Next Generation Sequencing (NGS) is a high-throughput method that enables highly parallel sequencing of multiple libraries based on prepared DNA or RNA samples. Today, applications such as genome-wide, all-exon and gene expression analysis are growing rapidly, and NGS is becoming more and more important and has become the standard procedure in many laboratories.
Results and Discussion
Figure 3: Standard curves of manual (A) and automated (B) qPCR setups, as well as a summary of associated data (C).
Figure 4: Comparison between manual and automated serial dilutions using Standard 0.
Figure 5: Comparison of automated serial dilutions for two NGS libraries.