The liquid biopsy revolution and its challenges

liquid biopsy cfDNA

When cells become apoptotic, their DNA gets fragmented to a distinct size of 160–180 bp and released into the bloodstream. These cell-free DNAs remain for some time as circulating fragments in the blood and – similarly to other blood analytes – can be assessed through a simple blood draw. As apoptosis is a normal cellular process that all cells in the human body eventually undergo, isolating and analyzing these cell-free DNA fragments from a blood sample can provide a comprehensive picture of the mutational profile of cells across the human body.

However, the analysis of cell-free DNA requires sensitive and comprehensive high-throughput methods to capture the full complexity and diversity of cell-free DNA molecules. Such analysis tools have only become broadly available and affordable with the advent of Next-Generation Sequencing (NGS) methods in the last 5–8 years.

Tumor cells in a cancer patient undergo exactly the same mechanism of releasing their DNA to the bloodstream when they become apoptotic. Thus, the cell-free DNA obtained from a blood draw of a cancer patient contains not only normal cell-free DNA molecules, but also tumor-specific molecules which resemble the complete tumor genome (1) and provide a view on the tumor-specific mutational profile. The analysis of cell-free DNA from a cancer patient to get specific information about the tumor is referred to as a “Liquid Biopsy” and is an especially promising technique for advanced tumor cases where invasive approaches to obtain tissue biopsy are life-threatening to the patient or simply not possible.

Since this potential has been embraced by the scientific and clinical community, a lot of research effort has been put into better understanding the molecular mechanisms and properties of cell-free DNA and has generated a lot of exciting findings around cell-free DNA in the last couple of years – even going towards methylation profiling of cell-free DNA to investigate the tissue origin of circulating DNA. Methylation profiling of ccfDNA can further hint at the origin of tumor-specific aberrations (2) and can show tumor-specific methylation patterns, e.g. at transcription start sites (3).

Cell-free DNA remains a challenging analyte

Despite the strong potential of cell-free DNA as a source for biomarker discoveries, it remains a challenging analyte. ccfDNA is highly variable in the plasma and varies not only from person to person, but also depending on the disease status (4). Amounts of cell-free DNA in plasma are usually limited with 1–100 ng/ ml plasma and additionally the signal-to-noise ratio between circulating tumor DNA fragments and normal circulating DNA is low. Cell-free DNA fragments are also fairly small with a mean size of 160–180 bp and require specific extraction and NGS library size selection protocols.

Another aspect about ccfDNA analysis is the accurate quantification of tumor-derived vs. normal DNA fragments. The amount of circulating tumor DNA in the blood can be directly linked to tumor burden, cancer therapy success and overall prognosis (3). Thus, accurate quantification of ccfDNA is essential to accurately monitor a cancer patient with a liquid biopsy.

Considering these points, dedicated solutions are needed for the analysis of cell-free DNA to maximize yields, capture the full sample complexity and obtain highly-sensitive and reliable results.

Learn more about the dedicated solutions QIAGEN offers in the field of Liquid Biopsy at

Are you specifically looking for a solution to go from plasma to NGS-ready library? Then the QIAseq cfDNA Library Kit is the right solution for you.

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Interested in knowing more about ccfDNA? Check out the recordings for our ccfDNA webinar series!

Part 1: Less is more – improved methods for fast and efficient ccfDNA purification using spin-column-based methods (Learn more)

Part 2: Standardize and streamline ccfDNA-based liquid biopsy workflows through automation using the QIAsymphony SP (Learn more)

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Verena Schramm

Dr. Verena Schramm is a Global Product Manager for NGS library preparation products at QIAGEN. She holds a degree in Molecular Biology and Bioinformatics from the University of Applied Sciences, Gelsenkirchen and has worked on various NGS-related applications in industry and academia since 2008. After completing her PhD at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany in 2014 and prior to joining QIAGEN, she worked for a Swiss start-up in the sector of Data Driven Medicine.

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