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Cell-free DNA Analysis for Sensitive Downstream Applications

cfDNA Analysis with the Agilent Automated Electrophoresis portfolio

What is cfDNA?

In eukaryotes, genomic DNA (gDNA) wraps around histone proteins, forming nucleosomes. Several nucleosomes come together to form euchromatin. During the process of apoptosis, endogenous endonucleases cleave DNA in repeatable lengths, based on nucleosome positioning. Cell-free DNA (cfDNA) is composed of the length of DNA wrapped around the nucleosome, the different lengths of DNA linking the nucleosomes, and degradation of the linkers between the nucleosomes (Figure 1). Thus, electrophoretic separations of cfDNA often display three fragments: the mono-nucleosome (~165 bp), di-nucleosome (~350 bp), and tri-nucleosome fragments (~565 bp). However, each sample is different, and may display anywhere from one to six or more fragment sizes. cfDNA can be extracted from many different bodily fluids, providing a valuable non-invasive resource for genetic information.

Representative image of cfDNA
Representative image of cfDNA, with a strand of DNA wrapped around a series of nucleosomes.

To accurately and reliably quantify, size, and qualify cfDNA for sensitive downstream applications, high detection sensitivity and separation by size is required. For example, continuous improvements to NGS technologies have enabled the sequencing of cfDNA. Successful sequencing depends on multiple quality control (QC) checkpoints to provide high-quality NGS libraries. cfDNA samples must undergo analysis to ensure that they are of sufficient quality for sequencing. Agilent provides several solutions for cfDNA QC analysis with the Automated Electrophoresis portfolio. Several kits are available for the TapeStation, Fragment Analyzer, Femto Pulse, and Bioanalyzer systems that provide the requisite sensitivity and resolution to reliably quantify, size, and qualify cfDNA fragments. While these instruments are designed for the QC of nucleic acids, they each offer different advantages in terms of throughput, sensitivity, speed, and resolution.

TapeStation Systems

The Cell-free DNA ScreenTape assay for the TapeStation systems provides fast, automated, and reliable digital assessments of cell-free DNA samples, with the detection of high molecular weight DNA contaminations.

Fragment Analyzer
Systems

Several analysis kits with the Fragment Analyzer systems offer high resolution separations of the mono-, di-, and tri-nucleosome cfDNA fragments with accurate and reliable sizing and quantification.

Bioanalyzer System

The High Sensitivity DNA assay for the Bioanalyzer sytem allows for separation resolution of the three major cfDNA peaks, apart from any high molecular weight (HMW) DNA present in the sample.

Femto Pulse System

The Ultra Sensitivity NGS kit for the Femto Pulse system provides complete separation of fragments which enables the precise and dependable sizing and quantification required for comparison of cfDNA levels in screening assays.

cfDNA Analysis Kits

Several instrument offerings from Agilent provide effective solutions for the QC analysis of cfDNA. With the relevant reagent kits, the instruments provide unmatched sizing and quantification capabilities.

cfDNA Application Notes

Don't stop learning here, read more about cfDNA analysis in our application notes.

For Research Use Only. Not for use in diagnostic procedures.

TapeStation Systems

Cell-free DNA ScreenTape assay

The Cell-free DNA ScreenTape assay for the TapeStation systems provides fast, automated, and reliable digital assessments of cfDNA. The assay provides accurate sizing and quantification of cfDNA from 50 to 800 bp, with input concentrations as low as 20 pg/µl. Quantify the cfDNA independent of any high molecular weight (HMW) DNA contamination using a region analysis function. High molecular weight DNA contamination can affect downstream NGS library preparation and sequencing quality. A new quality metric, %cfDNA, qualifies cfDNA samples by reporting the percent of cfDNA components present in the preset region of 50 to 700 bp relative to the total sample. This metric allows the user to objectively qualify the cfDNA, based on high molecular weight DNA content, for downstream analysis. The assay is compatible with common cfDNA extraction kits, including those that use carrier RNA to enhance the DNA recovery. Ensure your cfDNA is suitable for its downstream purpose with the highly reliable and reproducible quality assessment delivered by the TapeStation systems and the Cell-free DNA ScreenTape assay.

Electropherogram overlay of cfDNA samples with differing sample quality. Sample A (green, 72 %cfDNA) shows low abundance of HMW material >700 bp, reflecting the highest quality of the sample set. Sample B (orange, 57 %cfDNA) has a lower quality because it contains a higher amount of HMW material. Sample C (red) displays the lowest quality of 41 %cfDNA, due to a significant amount of HMW material.

cfDNA Samples on the TapeStation system

For Research Use Only. Not for use in diagnostic procedures.

Fragment Analyzer Systems

HS Small Fragment kit

The HS Small Fragment kit (p/n DNF-477) with a Fragment Analyzer system confidently sizes and quantifies the mono-, di-, and tri-nucleosome cfDNA fragments with high separation resolution. The small sizing range from 50 to 1,500 bp is perfect for reliable sizing and quantification of the small cfDNA fragments. However, it does not allow for complete separation of the tri-nucleosome cfDNA fragment from smaller fragmented genomic DNA (gDNA). Incomplete gDNA separation can affect the total cfDNA concentration and possibly sizing of the tri-nucleosome fragment. Carrier RNA occasionally used in cfDNA extraction does not interfere with cfDNA analysis on the HS Small Fragment kit allowing for accurate quantification of the total cfDNA sample.

cfDNA separated with the HS Small Fragment kit. Capillary electrophoresis performed on a Fragment Analyzer system equipped with a short capillary array, 33 cm.

cfDNA separations on the Fragment Analyzer system with the HS Small Fragment kit

HS NGS Fragment kit

The HS NGS Fragment kit (p/n DNF-474) with the Fragment Analyzer system offers high resolution separation of the three cfDNA fragments with accurate and reliable sizing and quantification. This reagent kit covers a size range larger than the cfDNA fragments allowing for separation from any fragmented gDNA. Complete separation from extraneous DNA allows for accurate quantification that is essential when cfDNA levels are utilized as a screening tool.

Some cfDNA extraction reagent kits utilize carrier RNA to help improve the isolation of cfDNA. The carrier RNA can be detected with the HS NGS Fragment kit and separates with the di-nucleosome cfDNA peak. When using this kit, it is recommended to avoid carrier RNA, or treat samples with RNase, to allow for complete separation of all cfDNA peaks and achieve reliable sizing and quantification.

cfDNA separations with the HS NGS Fragment kit (p/n DNF-474) with the Fragment Analyzer

cfDNA with fragmented DNA separated with the HS NGS Fragment kit. Capillary electrophoresis performed on a Fragment Analyzer system equipped with a short capillary array, 33 cm.

HS Large Fragment 50 kb kit

The HS Large Fragment 50 kb kit (p/n DNF-464) with a Fragment Analyzer system offers complete separation of all three common cfDNA fragments. With a large sizing range of 75 through 50,000 bp, accurate and reliable sizing and quantification of each fragment is achieved. An advantage of the wide sizing range is complete separation of fragmented DNA from the cfDNA fragments, allowing for accurate quantification of the total cfDNA sample using smear analysis. The HS Large Fragment 50 kb kit also allows visualization of any possible cfDNA fragments larger than the tri-nucleosome fragment. Carrier RNA occasionally used in cfDNA extraction can interfere with cfDNA analysis on the HS Large Fragment 50 kb kit, causing a small smear that overlaps with the tri-nucleosome fragment. The HS Large Fragment 50 kb kit is recommended for use in the absence of carrier RNA to completely separate and analyze cfDNA separate from any gDNA contamination.

cfDNA with fragmented DNA separated with the HS Large Fragment 50 kb kit. Capillary electrophoresis performed on a Fragment Analyzer system equipped with a short capillary array, 33 cm.

cfDNA separations on the Fragment Analyzer system with the HS Large Fragment 50kb kit

For Research Use Only. Not for use in diagnostic procedures.

Bioanalyzer System

High Sensitivity DNA assay

With the Bioanalyzer system, use the High Sensitivity DNA assay for analysis for cfDNA. The resolution of this system allows for the separation of the three major cfDNA peaks, apart from any high molecular weight (HMW) DNA present in the sample. For yield and purity analysis of the cfDNA subcomponents, a region analysis allows you to determine the percent of the total sample that is cfDNA. cfDNA isolated from common extraction kits, including those that use carrier RNA, is compatible with the Bioanalyzer High Sensitivity DNA assay.

Cell-free DNA sample analyzed with the High Sensitivity DNA assay on a Bioanalyzer system. The region highlighted in the electropherogram with a smear range set from 50 to 700 bp (blue lines) indicates the mono-nucleosome, di-nucleosome, and tri-nucleosome peaks, separated from the high molecular weight DNA.

cfDNA separated on the Bioanalyzer system with the HS DNA assay

For Research Use Only. Not for use in diagnostic procedures.

Femto Pulse System

Ultra Sensitivity NGS Kit

The Ultra Sensitivity NGS kit (p/n FP-1101) for the Femto Pulse system offers separation of low concentration smear levels, with an input concentration range of 5 pg/µL to 250 pg/µL. Sample conservation is an asset for precious and low concentration samples. The reagent kit covers a broad sizing range from 100 to 6,000 bp that enables complete separation of cfDNA fragments. The Ultra Sensitivity NGS kit can separate seven cfDNA fragments down to 62.5 pg/µL, with complete separation of the first three fragments down to 7.8 pg/µL. Complete separation of fragments enables the precise and dependable sizing and quantification required for comparison of cfDNA levels in screening assays. It is recommended to avoid carrier RNA, or treat samples with RNase, when analyzing samples with this kit to allow for complete separation of all cfDNA peaks and achieve reliable sizing and quantification.

cfDNA at 62.5 pg/µL separated with the Ultra Sensitivity NGS kit
cfDNA at 62.5 pg/µL separated with the Ultra Sensitivity NGS kit. Capillary electrophoresis performed on a Femto Pulse system.

cfDNA at 7.8 pg/µL separated with the Ultra Sensitivity NGS kit
cfDNA at 7.8 pg/µL separated with the Ultra Sensitivity NGS kit. Capillary electrophoresis performed on a Femto Pulse system.

For Research Use Only. Not for use in diagnostic procedures.

cfDNA Analysis Kits

The automated electrophoresis instruments provided by Agilent provide accurate and versatile analysis of DNA and RNA samples. The TapeStation, Fragment Analyzer, Bioanalyzer, and Femto Pulse systems provide variable throughput, high sensitivity detection, and broad sizing ranges for trusted nucleic acid QC.

Instrument Part Number(s) Reagent Kit Name Sizing Range Qualitative Range
TapeStation systems 5067-5630 (ScreenTape)
5067-5631 (Reagents)
Cell-free DNA ScreenTape 50 to 800 bp 100 to 4,000 pg/µL
%cfDNA functional: 100 to 5,000 pg/µL1
Fragment Analyzer systems DNF-477-0500
DNF-477-1000
HS Small Fragment kit 50 to 1,500 bp 100 to 5,000 pg/µL2
DNF-474-0500
DNF-474-1000
HS NGS Fragment kit 100 to 6,000 bp 50 to 5,000 pg/µL2
DNF-464-0500 HS Large Fragment 50 kb kit 75 to 50,000 bp 50 to 5,000 pg/µL2
Bioanalyzer system 5067–4626 High Sensitivity DNA kit 50 to 7,000 bp 100 to 10,000 pg/µL2
Femto Pulse system FP-1101-0275 Ultra Sensitivity NGS kit 100 to 6,000 bp 25 to 250 pg/µL2
Detection: 5 to 250 pg/µL1,2

1 Qualitative detection range
2 Concentration range determined with DNA smears

For Research Use Only. Not for use in diagnostic procedures.

cfDNA Application Notes

Performance Characteristics of the Cell-free DNA ScreenTape Assay

The Agilent Cell-free DNA ScreenTape assay was developed for cfDNA analysis, providing reliable total DNA concentration as well as quantitative assessment of cfDNA subcomponents apart from HMW DNA.

Separation of cfDNA with the Agilent HS NGS Kit on the Agilent Fragment Analyzer Systems

The Agilent 5200 Fragment Analyzer system offers excellent resolution and reliable sizing of circulating cell-free DNA (cfDNA).

cfDNA Separated on the Agilent Femto Pulse System

The Agilent Femto Pulse system and the Ultra-Sensitivity NGS kit offers unparalleled resolution and quality assessment of low concentrated cfDNA samples

Accurate QC Analysis of cfDNA Using the Agilent Fragment Analyzer System

This application note compares cfDNA peak separation using two reagent kits and two different capillary arrays for the Agilent Fragment Analyzer system.

For Research Use Only. Not for use in diagnostic procedures.