Agilent Nucleic Acid Analysis User Manual

Automated Parallel Capillary Electrophoresis

Nucleic Acid Analysis for Sample Quality Assessment Using the Agilent Fragment Analyzer Systems

Application Compendium

Agilent Automated Electrophoresis

Nucleic Acid Analysis for Sample Quality Assessment Using the Agilent Fragment Analyzer Systems

Application Compendium

Contents

Nucleic Acid Analysis for Sample Quality Assessment Using the Agilent Fragment Analyzer Systems 4

Product Information

Application Notes and Technical Overviews

Agilent Fragment Analyzer Systems 6

Capillary Electrophoresis 6

Fragment Analyzer Models 7

Benefits of the Fragment Analyzer systems 7 Reagent Kits for the Fragment Analyzer systems 8

Quality Metrics Overview 10

Genomic DNA quality number (GQN) 11 RNA quality number (RQN) 11 DV

200

Analysis of DNA 12

GQN quality metrics for genomic DNA quality assessment 12 Best sizing practices 13 Best quantification practices 14 Separation resolution of fragments 15 Consistent sizing of genomic DNA 16

Next-Generation Sequencing (NGS) 17

Library size and quantification comparison with two kits 17 Quality control in the Agilent Magnis SureSelect XT HS workflow 18 Library comparison between the Fragment Analyzer and Bioanalyzer systems 19

Analysis of Cell-free DNA (cfDNA) 20

Quality control analysis of cfDNA 20 Separation of cfDNA with the HS NGS Fragment kit 21

Analysis of RNA 22

RQN quality metrics 22 Eukaryotic total RNA 23 Comparison of the RIN and RQN 24 Assessing quality of plant RNA 25

Analysis of IVT mRNA 26

Quality control of IVT mRNA 26 Long IVT mRNA sizing 27

CRISPR/Cas Engineering 28

Single-guide RNA quality assessment 28 Analysis of long, single-stranded DNA 29 Polyadenylation of Cas9 mRNA 30 Engineered restriction site to estimate CRISPR homology-directed repair efficiency 31

Nucleic Acid Analysis for Sample Quality Assessment Using the Agilent Fragment Analyzer Systems

Recent years have seen the proliferation of genetic and genomic analyses throughout both the research lab and the clinic. The applications of these technologies are as numerous as their methods and include markets such as:

– Pharmaceutical/biopharmaceutical

– Genetic testing labs

– University or Institute core facilities

– Sequencing service providers

– Biobanks and repositories

– Synthetic bio/genome engineering

No matter the application, getting the most from your research requires a robust workflow. The integration of sample assessment and quality control (QC) checkpoints into workflows helps accomplish this by providing accurate information about nucleic acid samples, aiding in workflow optimization and minimizing the use of unfit samples in downstream applications.

The Agilent Fragment Analyzer systems are parallel capillary electrophoresis instruments that were designed for reliable and accurate nucleic acid sample assessment. A broad range of application kits are available, allowing you to easily size, qualify, and quantify both DNA and RNA

– Plant, forestry and animal genomic

analysis

– Environmental testing

– Clinical research and human disease

research

– Microbial/infectious disease research

samples. With its unique design and intuitive features, common QC bottlenecks are eliminated, and lab efficiency is increased with the ability to load multiple gels for both DNA and RNA applications. There are three models to choose from with varying throughputs to match your lab’s needs.

Common analysis workflows and samples that leverage the Fragment Analyzer systems for quality assessment include:

– Genomic DNA (gDNA)

– Cell-free DNA (cfDNA)

– Formalin-fixed paraffin-embedded (FFPE)

– Plasmid DNA

– CRISPR-TILLING mutation detection

– CRISPR guide RNA

DNA

– Polyadenylated IVT RNA

– Next-generation sequencing (NGS) library

preparation

– Genotyping

– PCR amplicons

– Simple sequence repeats (SSR)/

microsatellite amplicons

– Total RNA

– mRNA

– IVT RNA

– FFPE RNA

– Small RNAs (including microRNAs)

– Restriction digest analysis

To demonstrate the benefits of sample QC in these workflows, we compiled this compendium of application notes written by Agilent scientists. We also briefly describe capillary electrophoresis technology and the Fragment Analyzer system product portfolio. This application compendium demonstrates the advantages offered by sample assessment and how easily it can be incorporated into virtually any genomics workflow.

Agilent Fragment Analyzer Systems

The Fragment Analyzer systems utilize automated parallel capillary electrophoresis to provide reliable QC for a broad variety of sample types and applications, including NGS libraries, RNA samples, PCR amplicons, and CRISPR workflows.

The Fragment Analyzer systems break through analytic bottlenecks by providing a sample quality assessment tool that easily integrates with key genetic analysis workflows, giving researchers the results they need, when they need them. Automated parallel capillary electrophoresis enables analysis of multiple samples at once without researcher intervention. The ability to accommodate two different gel matrices allows for unattended and consecutive analysis of multiple reagent kits.

The Fragment Analyzer systems are indispensable for sample QC because of their many benefits, including:

– Flexible, interchangeable arrays allow for adjustable throughputs and

applications to fit the changing needs of the laboratory

– Minimal sample concentration requirements enable researchers to conserve

precious samples for further analysis

Capillary electrophoresis

The capillary array is the basis of the Fragment Analyzer systems, as it can reliably separate both DNA and RNA samples, easily switching between applications. The arrays are available in three different lengths (short, ultrashort, and long) to allow the user to prioritize separation resolution or time, depending on workflow needs. A shorter capillary array offers faster run times, while longer capillary arrays offer improved separation resolution.

Principles of capillary electrophoresis:

1. Individual capillaries are filled with gel that serves as the separation matrix

2. Samples are voltage-injected into the capillaries, and each sample moves through an individual capillary in a size-dependent manner

3. As the fragments pass the detection window, a sensitive charged coupled device (CCD) detector captures the size and concentration level of the sample

4. The resulting series of images are merged together to produce a highresolution electropherogram across the entire sizing range

Fragment Analyzer Models

With three different models, the Fragment Analyzer systems are easily adaptable to the workflow of any lab:

The 5200 Fragment Analyzer and 5300 Fragment Analyzer systems provide variability in throughput by using different capillary arrays.

– The 5200 Fragment Analyzer system uses a 12-capillary array allowing for 12

samples to run at one time

– The 5300 Fragment Analyzer system offers higher throughput, with the ability

to run either a 48- or a 96-capillary array

– Both models have the ability to load up to three 96-well plates and process in

any order

The 5400 Fragment Analyzer system is an ultrahigh-throughput system, enabling the analysis of thousands of samples per day.

– The system utilizes a 96-capillary array and is capable of full integration with

most robotic systems

– An application program interface (API) controls the movement of the buffer,

waste, and sample drawers, allowing for continuous runs without user intervention

– Integrated software packages allow for remote control of the instrument

Note: Experiments performed using the 5200 Fragment Analyzer system and can be replicated

with comparable results on Agilent 5300 Fragment Analyzer and 5400 Fragment Analyzer systems when using the same array length.

Features and benefits of the Fragment Analyzer systems

– With electrophoresis times as short as 15 minutes, no daily array handling,

and room-temperature stable reagents, instrument set-up and run time is minimized allowing faster time to results

– 3 bp resolution of fragments approximately 300 bp and smaller provides

discrimination of closely sized products giving you confidence in sample composition

– Two orders of magnitude dynamic range enables loading of unknown

quantities, eliminating the need to predetermine sample concentration

– Quality metrics for RNA (RNA quality number, RQN) and genomic DNA

(genomic DNA quality number, GQN) allow for objective quality assessment aiding nucleic acid sample standardization

– Always-accessible sample drawers permit the addition of samples, even while

the instrument is running, eliminating down time

– Ability to re-arrange the sample queue even while the instrument is running

lets high priority samples to be run sooner

Reagent kits for the Fragment Analyzer systems

A broad range of kits are available for the Fragment Analyzer systems, allowing you to easily qualify and quantify both DNA and RNA samples. The diversity of sample types these systems can separate make these instruments ideal for a variety of workflows, including sample QC for NGS library preparation.

The Small Fragment and NGS kits facilitate the separation of DNA fragments, smears and NGS libraries, with sizing from 50 to 1,500 bp and 100 to 6,000 bp, respectively. The NGS kit covers a concentration range of 0.1 to 10 ng/µL for fragments and 5 to 100 ng/µL for smears. The HS Small Fragment and HS NGS kits have a smaller concentration range of 5 to 500 pg/µL for fragments and 50 to 5,000 pg/µL for smears. Each of the kits provides accurate quantification and sizing, making them ideal for NGS library preparation workflows.

The Large Fragment kits are used for automated qualitative and quantitative analysis for large DNA fragments and smear up to 48,500 kb. Each of the kits has a different input concentration range, specific for fragments or smears. The Large Fragment kits are ideal for reliable QC checkpoints in many different workflows, including long-read NGS, formalin-fixed paraffin-embedded (FFPE) DNA analysis, and restriction digest analysis. By comparing the sizing of sheared and unsheared large DNA samples, the kits provide a reliable evaluation of DNA fragmentation, an essential step in the preparation of large-insert libraries.

The Genomic DNA kits were developed for the separation of genomic DNA (gDNA). Automated assessment of gDNA size and integrity is extremely beneficial for QC of samples to be used in long-read and whole genome NGS, metagenomics, and analysis of degraded DNA. A broad sizing range allows accurate and precise sizing of samples through 60 kb. Covering expansive concentration ranges, the HS gDNA 50 kb kit is for samples ranging from 0.3 to 12 ng/µL, while the gDNA 50 kb kit extends from 25 to 250 ng/µL. Easily analyze gDNA samples with a user-defined quality metric (GQN), which allows users to decide what qualifies as good gDNA for their purposes.

The RNA kits can be used for analysis of both total RNA and mRNA, including IVT mRNA sizing. Ensuring quality RNA is crucial to many downstream applications, including NGS and gene expression studies. RNA analysis with the RNA kits provides each sample a quality metric, the RQN. Excellent resolution allows for distinction between small RNA and degraded RNA, providing a reliable and accurate RQN score. Specific RNA analysis modes are available for eukaryotic, prokaryotic, plant, and mRNA samples. The RNA kits have a sizing range of 200

to 6,000 nt. The RNA kit (15 nt) covers a concentration range of 5 to 500 ng/μL,

while the HS RNA kit is for less concentrated samples, with a range of

50 to 5,000 pg/μL.

The Small RNA kits focus on microRNA and small RNA QC analysis, essential for downstream applications such as small RNA NGS library preparation. The kits provide accurate and precise quantification and sizing of small RNA and microRNA, focusing on the narrow range from 15 to 200 nt. Focusing on this small sizing range allows for high-resolution separation and detailed analysis of both the microRNA (10 to 40 nt) and small RNA (40 to 200 nt) regions. A region analysis function automatically calculates the percent microRNA and quantifies the microRNA and small RNA regions.

The Qualitative DNA kits provide automated and accurate sizing with relative quantification of DNA fragments. Common uses include the analysis of PCR amplicons, simple sequence repeats (SSRs)/microsatellites, and genotyping. There are six different kits to choose from based on the size of the DNA or PCR fragments, ranging from 35 to 20,000 bp. Specially formulated gels, markers, and ladders are designed for each kit, enabling reliable sizing over a broad range with varying applications. High-resolution kits enable the separation of fragments under 300 bp with a 3 bp difference.

The Plasmid DNA kit is used in the analysis of supercoiled and linear plasmid DNA. The ladder provided with this kit is optimized for the accurate sizing and relative quantification of supercoiled plasmids between 2,000 and 10,000 bp. Linearized plasmids can be evaluated for quality and relative quantification; however, only comparative sizing is possible.

Efficient and accurate identification of CRISPR mutation events is a critical component of CRISPR/Cas9 workflows. The CRISPR Discovery Gel kit automates the screening of heteroduplex cleavage assays and provides fragment sizing and relative concentration. These important pieces of information are automatically processed by ProSize data analysis software to indicate successful editing events and other crucial information like percent mutated.

Quality Metrics Overview

High-quality nucleic acids are necessary for successful library preparations and sequencing results. Nucleic acid QC can determine which samples are not suitable for library preparation. Not all extraction methods are the same, resulting in nucleic acids with varying integrity. Nucleic acid samples such as FFPE, ancient samples, and RNA are easily degraded due to chemical fixation, time, temperature, enzyme digestion, and improper handling. If a sample is too far degraded, it will result in poor sequencing results; important coding areas of interest are lost, and gaps appear in the full-length gDNA. Knowing the quality of the input nucleic acid helps provide guidance for changes needed to optimize workflow for applications such as NGS, long read sequencing, microarray, and genotyping.

Quality metrics provide the user with reliable assessment of the integrity of a sample. Users can establish quality metric standards in their workflows by doing degradation studies and comparing the quality metrics scores between the samples. In addition, quality metrics can save time and money by reducing variation between user assessments and misinterpretation of the results, while easily identifying unfit starting material that would lead to poor results. The Fragment Analyzer instruments provide reliable quality metrics for different sample types. The Agilent quality metrics are widely accepted and referenced in literature. The Fragment Analyzer systems ProSize data analysis software has the quality metrics built in for easy assessment of samples.

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