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When treating cancer, sooner is always better. As tumors mature, they grow more genetically diverse and difficult to knock down. By the time an aggressive cancer becomes symptomatic, it may be too late. That’s why research into liquid biopsies has grown in prevalence: these sample types have tremendous potential to facilitate early cancer detection and support ongoing surveillance of tumor evolution.

The value of liquid biopsies largely stems from the presence of cell free DNA (cfDNA) in bodily fluids. Healthy cells naturally shed fragments of DNA into the extracellular space, some of which will be swept up into circulation. These bits of cfDNA may include telling mutations or epigenetic patterns that can be used to not only trace the fragment back to its tissue of origin, but to learn about the genetic health of the tissue. The same holds true when that DNA is leaked from a budding cancer cell.

In the future, liquid biopsies may be used to detect fragments of circulating tumor DNA (ctDNA) and discover the existence of a nascent or recurrent tumor. However, considerable research will be needed to reach this stage. ctDNA is only a tiny part of any liquid biopsy, making this a true needle-in-a-haystack operation. A typical blood sample contains about 1 to 10 ng/ml of cfDNA, and ctDNA may only be about 1-10% of that (for small or poorly vascularized tumors, this number may drop to 0.01%). To help researchers expand our knowledge of ctDNA and its potential future value in clinical settings, Twist Bioscience has developed advanced target enrichment panels, reference standards, library preparation kits, and many other tools that are each designed to help researchers better capture and study ctDNA.

Click here to learn more about Twist products used in cancer research 

The Critical Need for Target Enrichment

If ctDNA is the needle in the haystack, target enrichment is the magnet that grabs it. Tagged oligo probes are designed to catch fragments of cfDNA that are likely to be harboring tumor-associated mutations (specifically targeting genomic sequences where malignant mutations are known to occur), while ignoring all other superfluous cfDNA fragments. Isolating these molecules focuses the search, reducing the need for sequencing and the associated costs.

While the concept of enrichment is simple, achieving accurate and sensitive enrichment is another story. Such a task requires careful probe design (particularly if the goal is to detect methylation marks), as well as accurate and uniform probe synthesis.

One way labs can improve their odds of success is to design a custom, tumor-informed enrichment panel. To do this, labs must build a mutational profile of the tumor being studied. Twist developed several target enrichment panels to enable efficient whole exome sequencing of tumor samples, even those stored in paraffin blocks after formalin fixation. Twist’s Human Core Exome and Exome 2.0, for example, provide comprehensive coverage over portions of the genome where tumor-associated mutations are likely to reside (primarily protein coding and other highly studied portions of DNA). Such data can be used to identify the tumor’s unique variant profile and inform the construction of a corresponding, custom ctDNA panel. Twist also offers custom target enrichment panels, designed specifically for ctDNA and minimal residual disease research, such as the MRD Rapid 500 Panel.

Whether using a custom, tumor-informed panel or an optimized off-the-shelf panel, controls are critical. To this end, Twist’s cfDNA Pan-Cancer Reference Standard v2 provides labs with reference material composed of wild-type, cell-line cfDNA and synthetic oligos containing mutant alleles. The kit includes over 400 circulating ctDNA variants, spanning 84 genes, mirroring cfDNA fragment size and distribution to simulate real-world biology. In doing so, this reference material enables characterization of NGS-based assays—including limits of detection and limits of blank—ultimately aiding the development of highly precise and sensitive liquid biopsy workflows.

The Crucial Need For Preparation

Library prep is an essential step in any next-generation sequencing (NGS) workflow, wherein genetic material is converted over to a format that’s compatible with high-throughput NGS. This may involve fragmentation, end repair, ligation of unique molecular identifiers and NGS adaptors, and finally amplification. Errors can occur at every step, each with the potential to reduce assay performance considerably.

Consider the rarity of ctDNA molecules in a liquid biopsy. Levels of ctDNA can dip to just 0.01% of the total cfDNA in a given sample. If library preparation fails to properly convert any of the few ctDNA molecules that are present, the assay’s sensitivity will likely drop significantly.

To enable improved library preparation, Twist has developed several kits with varying applications.

The Twist Library Preparation Enzymatic Fragmentation Kit 2.0 is well suited for processing of tumor samples thanks to the incorporation of new fragmentation and ligation enzymes. Additionally, the inclusion of the Equinox Library Prep Amp Mix, a hot-start enzyme formulation with a low error rate and high efficiency for low-volume inputs—makes it a powerful option for use in either tumor profiling or in the detection of ctDNA.

In addition, the Twist cfDNA Library Preparation Kit is designed specifically to enable sensitive liquid biopsy research. This kit is optimized for high-conversion of cfDNA libraries and has demonstrated the ability to detect variant alleles with frequencies as low as 0.1%. The kit is also suitable for duplex sequencing, further empowering researchers to develop robust and sensitive workflows.

Looking Beyond The Nucleotides

Tumor cells develop cancer-specific methylation patterns, activating genes that help them divide rapidly and survive. As a result, cfDNA provides a unique window into cancer development. By identifying methylation markers in cfDNA, labs may be able to detect malignant signatures that not only indicate a tumor’s presence, but also point to its tissue of origin.

To enable researchers interested in studying these methylation signatures, Twist has developed custom methylation panels and the Twist NGS Methylation Detection System. This system uses an enzymatic conversion process that minimizes DNA damage and loss during library preparation. Methylation conversion can be paired with Twist hybrid-capture off-the-shelf panels or ultra-precise custom panels, which capture methylated, unmethylated, sense, and antisense DNA. (You can read more about these tools here: Tools to Improve Methyl-seq Efficiency for Better Resolution in Epigenetic Studies)

Similarly, Twist’s Human Methylome Panel is designed to capture methylated bases across the genome, encompassing more than 84% of CpG islands. The panel targets 3.98M CpG sites, through 123 Mb, to find important methylation markers. The workflow employs a unique enzymatic process that is much less damaging to DNA. The panel is optimized for the Twist methylation detection system to provide a seamless workflow.

ctDNA—and liquid biopsies by extension—is poised to be a powerful tool in both research and clinical applications. Twist is committed to empowering this work, providing researchers with advanced tools and a team of experts to rely on. Detecting ctDNA may be tough, but with the right partners and tools, it gets a whole lot easier!