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AUTOMATED QC ANALYSIS FOR NGS LIBRARY PREPARATION AND CRISPR/CAS9 GENE EDITING
제니어 조회수:721 106.252.50.10
2017-01-09 13:26:13

 

AUTOMATED QC ANALYSIS FOR NGS LIBRARY PREPARATION AND CRISPR/CAS9 GENE EDITING

 

 

 

 

 

INTRODUCTION

Reliable analysis of the quality and quantity of DNA/RNA fragments and smears is essential for downstream applications such as next generation sequencing (NGS), genotyping, CRISPR/Cas9 gene editing and integrity assessment of genomic DNA, small RNA and total RNA. QC is recommended at multiple steps during the NGS library preparation process, typically after shearing or fragmentation, after adapter ligation and after library enrichment. Accurate determination of both the quality and quantity of the final library preparation is critical for molarity calculations and proper flow cell loading.

Traditionally, quality analysis of DNA fragments is performed by agarose gel electrophoresis, a method that is time consuming and labor intensive. The Fragment Analyzer INFINITY™ Automated CE System (FAI), manufactured by Advanced Analytical, is a multichannel capillary electrophoresis instrument offering high resolution separation of 96 samples in parallel. It can accurately size and quantify nucleic acid fragments and smears from 50bp to 50kb, and can hold two different gel reagents for seamless switching between applications and sample types. Over 20 reagent kits are available – each optimized for the analysis of different fragment lengths – to cover a wide range of analyses, including:

• NGS library preparations

• Large fragment DNA smears

• Genomic DNA

• small and microRNA

The powerful PROSize ® Data Analysis Software automatically assesses nucleic acids for fragment size, concentration and quality, assigning samples to different protocols accordingly. The accuracy and precision of the FAI sizing data is unmatched, at 5 % and 2 % respectively. Figure 1 shows a typical electropherogram produced from a standard library preparation.

Integration of the FAI onto the Freedom EVO ® workstation allows full automation of important quality control checkpoints for applications, such as NGS sample preparation and CRISPR/Cas9 gene editing workflows.

Figure 1: Dilution series analysis of an NGS library preparation from genomic DNA. Samples were analyzed with the FAI using the High Sensitivity NGS Fragment Analysis Kit (DNF-474). The average size of these library smears is 446bp, and was measured at three sample concentrations: 5 ng/µl (black line), 2.5 ng/µl (blue line) to 0.5 ng/µl (red line).

 

FRAGMENT ANALYZER INFINITY™

The FAI is a leading solution for automated analysis of the size, quality and quantity of nucleic acids. Controlled through a detailed and robust Application Program Interface (API), it is configured for seamless and continuous operation.

Each drawer on the FAI can be independently accessed by the Freedom EVO's Robotic Manipulator (RoMa) Arm, with buffers and waste automatically removed to allow truly continuous processing.

Fully integrated software for both operation and analysis allows users to remotely control electrophoresis separations, and all commands can be activated through either TCP/Ethernet or serial port connections. All errors are documented and logged to assist with troubleshooting, and data can be automatically analyzed and reported in a user defined format.

Figure 2 and 3: Integration of the FAI onto the right side of the Freedom EVO allows the RoMa Arm to automatically load and unload plates.

 

HARDWARE INTEGRATION

The FAI can be integrated, through the Tecan Integration Group (TIG), onto the right side of a Freedom EVO 100, 150 or 200, and accessed by the workstation's RoMa Arm (Figures 2 and 3). This configuration provides easy access to the FAI side panel to refill gels and conditioning liquids, as well as to empty the waste bottle.

 

DATA ANALYSIS SOFTWARE

PROSize Data Analysis Software simplifies fragment identification and analysis. This intuitive software package offers a number of unique features:

• Smear analysis providing concentration, molarity, size range, average size and %CV

• Flagging features to identify individual fragments or fragment combinations

• Quality (integrity) scoring for RNA, DNA, microRNA and FFPE-extracted nucleic acids

• Sample overlay with the ability to combine data from up to 24 different runs

• Customizable reports in PDF or CSV formats

 

SUMMARY

Integration of the FAI onto a Freedom EVO liquid handling platform enables incorporation of important quality control checkpoints into automated genomic workflows. This allows longer walkaway times and ensures that DNA fragments meet the quality control criteria necessary for subsequent sequencing or other downstream analysis.

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