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Circulating, Cell-Free DNA

Circulating, cell-free nucleic acids have emerged as important biomarkers in cancer and clinical diagnostics and detection of various clinical conditions. Translational genomic research is utilizing cell-free DNA (cfDNA) to monitor personalized therapies based on specific tumor profiles. In order to research this valuable information, it is critical to be able to detect the low levels of cfDNA from whole blood samples.


Accel-NGS 2S PCR-Free DNA Library Kit

The Swift advantage:
  • PCR-free libraries from 10 ng of cfDNA are compatible with limited input quantities.
  • High complexity libraries from less input enables deep sequencing for variant calling.
  • Minimal bias for AT-/GC-rich sequences provides accurate representation of inherently biased cfDNA.

Accel-Amplicon™ Panels

The Swift advantage:
  • 10 ng of input DNA required.
  • Amplicon sizes 120-160 bp are compatible with 165 bp cfDNA fragment sizes.
  • High coverage uniformity reads are evenly distributed across targets.

Whole Genome Sequencing of cfDNA

PCR-free Libraries from 10 ng Cell-Free Plasma DNA

The Accel-NGS® 2S PCR-Free DNA Library Kit is validated to make PCR-free libraries from 10 ng of cfDNA. Low input PCR-free library construction is enabled by the relatively undamaged ends of cfDNA and the narrow size distribution of cfDNA fragments.

Enzymatic fragmentation of cfDNA in the blood results in end bases that are much less damaged than those resulting from fragmentation by mechanical shearing. For mechanically sheared DNA, the 5’ and 3’ end repair of Accel-NGS 2S PCR-Free is capable of alleviating some end damage. However, irreparably damaged fragment ends prevent adapter attachment, and are not recovered in the final library. Therefore, the relatively undamaged ends of cfDNA are more likely to be constructed into library molecules, resulting in a higher conversion efficiency of input DNA into functional library molecules.

The narrow size distribution of cfDNA also contributes to increased conversion efficiency. The relatively wide size distribution of fragments created by mechanical or enzymatic shearing during library preparation results in loss of input DNA during size selection, whereas the inherently narrow size distribution of cfDNA centering around 165 bp reduces the loss of input DNA. Together, Swift delivers conversion rates of well over 60% and can be as high as 90% with the Accel-NGS 2S PCR-Free DNA Library Kit.

Increased cfDNA Conversion Efficiency Enables 10 ng PCR-free Library Construction

PCR-Free Libraries from 10 ng cfDNA

AP_cfDNA_Table_PCR-Free Libraries

cfDNA Insert Size


The minimal sequence-dependent bias of Accel-NGS 2S PCR-Free adapter attachment results in libraries that faithfully represent the cfDNA sample. The Accel-NGS 2S PCR-Free library coverage plot below illustrates the inherent loss of AT-rich sequences in cfDNA. This bias is not observed with mechanically sheared human DNA samples.

Coverage of Inherently Biased cfDNA Sample


 Targeted Sequencing of Circulating, Cell-Free DNA

Accel-Amplicon™ Panels with cfDNA

Accel-Amplicon Panels for Illumina® can be used with cfDNA samples to screen for clinically relevant mutations. Primer pairs within the panels are designed for an amplicon size range of 120-160 bp. For this reason the Accel-Amplicon Panels are well suited for samples containing short DNA fragments, such as FFPE and cfDNA. The fast, single-tube workflow of the Accel-Amplicon Kit requires just 10 ng of input DNA and provides high coverage.

Amplicon Coverage for gDNA, Fresh Frozen, FFPE and cfDNA Samples

AP_Amp Seq_Image_High Cov Uniform

Concentration and Integrity of cfDNA

To quantify your cfDNA and assess the amount of high molecular weight gDNA contamination in your cfDNA sample, we recommend utilizing primers designed for highly abundant ALU sequences in human genomic DNA to construct short (115 bp) and long (247 bp) ALU amplicons. Using this method, qPCR quantification of 115 bp ALU amplicons (ALU115) and 247 bp ALU amplicons (ALU247) corresponds to the concentration of total DNA (cfDNA and gDNA) and gDNA, respectively. The ALU247/ALU115 ratio indicates the DNA integrity index of the sample. ALU115-qPCR results can be compared to a standard curve to quantify cfDNA within the sample.