Cell Free DNA (cfDNA)

The emergence of liquid biopsies and non-invasive progression monitoring has resulted in the development of many new Cell Free DNA (cfDNA) assays.

What is a Liquid Biopsy?

A liquid biopsy enables sequencing of DNA in a few drops of a person’s blood or plasma. Unlike a traditional biopsy, a liquid biopsy is largely non-invasive and therefore is a convenient technique for diagnosing or monitoring diseases such as cancer.

Extraction of cell free nucleic acids from biofluids (e.g. blood, plasma & urine) can be challenging as they often yield very low quantities of highly-fragmented DNA. Assays for cell free DNA are intended to identify genetic variants present at very low allelic frequencies, as low as 0.1% and beyond, which challenges the detection limits of many current technologies.

Also detection of structural variations (gene translocations, fusions, copy number variants & large INDELs) are challenging and bioinformatics pipelines are continuously evolving to improve structural variant detection. Detection of SNVs and small INDELs in highly repetitive regions (high GC or AT content) is difficult as the depth of coverage in exome and targeted-capture data is particularly susceptible to GC bias, uneven coverage near the boundaries of the capture baits, and other systematic biases.

Profiling cfDNA

Cell Free DNA (cfDNA)

Horizon has developed Reference Standards to support the advancement and continued evaluation of cfDNA workflows across a range of platforms including NGS, ddPCR and qPCR. These high-throughput platforms give researchers greater power when profiling cfDNA samples, however further consideration must be given to sample variability, platform bias, and potential failure to detect biomarkers.

As a result it is important to have an independent assay control to enable researchers to prove the performance including sensitivity, specificity and accuracy of their cell free dna assays and platforms. Horizon's Reference Standards have been developed in response to this need.

cfDNA Reference Standards

These standards are manufactured from engineered human cancer cell lines, thereby providing a consistent and renewable resource for cell free DNA workflow evaluation. Reference Standards are fragmented to an average size of 160bp to resemble that extracted from human plasma (see Figure 1).

Figure 1. Example of fragment sizes of cfDNA Reference Standards collected by D1000 DNA ScreenTape assay.

Note: The left peaks, centered at 25 bp, represent an internal marker for the assay. The fragmented material is represented by larger, right peaks.

cfDNA Reference Material

Horizon cfDNA Reference Standards are available in several blends to address specific assay challenges including assessing assay specificity, challenging the limit of detection and confirming structural variant detection in cell free DNA workflows.

Cell Free DNA Products

Reference StandardDescriptionKey Applications
Multiplex I
Reference Standard
in Synthetic Plasma

Multiplex panel in synthetic plasma with 8 onco-relevant variants across 4 commonly mutated genes

In a set containing fragmented DNA in a range of low allelic frequencies (5%, 1% and 0.1%), with a matched wild type

Whole process control to validate and develop new ctDNA NGS and ddPCR assays at low allelic frequencies (down to 0.1% AF)
• Evaluate specificity and sensitivity of ctDNA assay
• Matched wild type to assess sensitivity of ctDNA assay
Multiplex I
Reference Standard

Multiplex panel with 8 onco-relevant variants across 4 commonly mutated genes

In a set containing fragmented DNA in a range of low allelic frequencies (5%, 1% and 0.1%), with a matched wild type

• Validate and develop new ctDNA NGS and ddPCR assays at low allelic frequencies (down to 0.1% AF)
• Evaluate specificity and sensitivity of ctDNA assay
• Matched wild type to assess sensitivity of ctDNA assay
BRAF V600e
Reference Standard

Gene-focused singleplex with a onco-relevant variant

In a set containing fragmented DNA in a range of low allelic frequencies (5%, 1% and 0.1%), with a matched wild type

• Validate and develop new ctDNA qPCR assays at low allelic frequencies (down to 0.1% AF)
• Matched wild type to assess sensitivity of ctDNA assay
• Generate standard curves to determined the limit of detection of ctDNA assay
Structural Multiplex
Reference Standard

Multiplex panel with 10 onco-relevant structural variants across 8 commonly mutated genes

Single tube containing fragmented DNA in allelic frequencies 4.8-5.6% and 4.5 - 9.8 x amplification for copy number variant (CNV)

• Validate and develop new ctDNA assays detecting structural variants
• Evaluate the effect of genomic context on variant detection.
• Analyse the robustness of your bioinformatics pipeline for structural variant detection

cfDNA Resources

Still have questions? Click one of the following links for more information:

A complete list of frequently asked questions about our cell free DNA Reference Standards

cfDNA FAQ

Analysis of circulating free DNA at a 1% variant allele fraction using the GeneReader™ NGS System

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Allelic Frequency Measurement of Multiplex I cfDNA Reference Standard Set using Droplet Digital PCR, Ion Torrent and MiSeq

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