August 1, 2023 | Genomics

Unfolding the cfDNA story: Q&A highlights from our webinar

Webinar Q&A insights that can help unravel the story of your cfDNA sample

A cfDNA sample could be hiding an ultra-rare variant that’s yet to be detected. Are your current tools and technologies sensitive enough to uncover this elusive variant? Unfold the story of your cfDNA sample with helpful tips from our recent webinar, “Striking gold: Finding ultra-rare variants in liquid biopsy samples using targeted NGS.” We received a lot of insightful questions from our highly engaged audience during the Q&A session. Check out the answers below and boost your knowledge.

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1. What is the typical ctDNA concentration in plasma or serum for cancer patients? 
What is the yield of ctDNA sample collection with the QIAamp Kit?

The concentration of circulating tumor DNA (ctDNA) in plasma or serum can vary widely among cancer patients and is influenced by various factors such as the tumor type, stage and treatment history. 

The typical concentration of ctDNA in plasma or serum can range from a few copies per milliliter (copies/mL) to several thousand copies/mL.

In early-stage cancers or cancers with low tumor burden, the concentration of ctDNA in plasma or serum may be relatively low, often in the range of 1–100 copies/mL. As the cancer progresses or the tumor burden increases, the concentration of ctDNA tends to rise, potentially reaching several hundred to several thousand copies/mL in advanced-stage or metastatic cancers.

The exact yield of ctDNA sample collection with the QIAamp Kit can be influenced by the quality of the starting material.

2. How long does it take to process samples and how accurate is the result?

With QIAseq Targeted cfDNA Ultra Panels, you can go from cfDNA sample to sequencing-ready library in just 8 hours. The entire workflow, from your sample collection to data analysis and interpretation, can take up to 4 days. 

3. Can you achieve low VAF without NGS?

Digital PCR technologies are great for detecting variants at low VAF if you know which variants you want to detect.

4. What is the importance of uncovering rare variants?

Tumor-derived cfDNA, including ctDNA, can carry rare variants specific to the tumor and serve as non-invasive cancer biomarkers. Identifying these rare variants can aid in early detection, monitoring treatment response and detecting minimal residual disease or recurrence.

5. How can I distinguish between actual variants and artifacts at low VAF values?

High-fidelity sample and library preparation can help lower artifacts. This means increased accuracy in detecting actual variants, resulting in high-confidence data.

You can also incorporate unique molecular indices (UMI) that allow you to distinguish real variants from any technical variants introduced during sample processing, such as artifacts due to PCR errors and bias. 

Post-sequencing, you can also put error-correction data analysis modules in place to filter out artifacts.

6. What is the least amount of DNA quantity required to study rare variants from cfDNA?

The minimum cfDNA input requirement is 10 ng – the more your input, the higher your sensitivity.

7. Is duplex sequencing available for this workflow?

Duplex sequencing is not available.

8. How can I design a targeted panel and sequencing experiment (coverage, probe density) to detect variants at 1% of VAF?

We do have several pre-designed panels available. These panels come with sequencing guidelines regarding coverage needed to achieve low VAFs. You can find this information in the kit handbook. Our Enterprise Genomics Solutions team can also work with you to design a custom panel. You can book a consultation here. Custom QIAseq Targeted cfDNA Ultra Panels will also be available soon.

9. Is the extraction technique a critical element in ensuring this level of testing precision?

The low concentration of cfDNA in non-advanced, non-metastatic cancers limits the available concentration as a testing analyte to well below 10 ng/µL. Scalable and efficient purification of high-quality cfDNA from serum and plasma samples is needed, even when small amounts are present.

10. Do you have custom solutions?

We currently offer booster options allowing you to add targets for up to 100 primers to our existing panel. We will enable customization for the panels for you to design complete custom panels with your target inputs in the near future.

11. How many gene regions can be targeted simultaneously and how does this impact the detection of variants at low VAF?

It depends. With QIAseq Targeted cfDNA Ultra Panels, you can target up to 20,000 primers, which is about 500 genes. The impact on detecting low VAF is that the larger your panel, the more you need to sequence and the more cfDNA input you need. It depends on your target VAF. For example, in the case of 1% VAF vs. 0.1%,  0.1% VAF will require more ctDNA/cfDNA and sequencing at a greater depth than 1% VAF.

12. What is the density tiling?

Primers are 100–150 bp apart to capture the full range of your cfDNA since it can fragment anywhere in the cfDNA molecule. The density is about two primers per region of interest.

13. How does the sensitivity of QIAseq Targeted cfDNA Ultra Panels compare to dPCR?

QIAseq Targeted cfDNA Ultra Panels target to 0.1% VAF, but dPCR can go down to 0.05% with the right input. The difference is that with dPCR, you need knowledge of your variants ahead of time, and NGS provides the discovery power, so you don’t need to have knowledge about the variants ahead of time.

14. How many unique DNA molecules do you sequence with 10 ng input?

Our capture efficiency is 30–50% for unique DNA molecules, enabling the detection of variants at 0.2% frequency.

15. What is the difference between the current panels (QIAseq Targeted DNA Panels) and the new technology (QIAseq Targeted cfDNA Ultra Panels) and what are the analysis options?

QIAseq Targeted DNA Panels use Taq DNA Polymerase during library prep and fragmentation. Anytime you fragment your DNA, you introduce background error. This limits your VAF to 0.5–1% or above. With the new technology introduced in QIAseq Targeted cfDNA Ultra Panels, we skip fragmentation and incorporate a high-fidelity polymerase that allows you to systematically lower the background error to detect variants at low VAF.

An optimized bioinformatics pipeline with CLC Genomics Workbench, set to detect 0.1% VAF at 99% specificity, is included for analysis. 

16. What is the recommended UMI depth for ctDNA detection?

The recommendation is based on the amount of ctDNA you use as input. We usually require about 40k x coverage, which gives you 4–8 UMIs per molecule, which provides enough sensitivity to detect 0.1% VAF. Ultimately, it depends on what sequencing depth you will utilize for VAF.

17. What is the least amount of DNA input needed to study cfDNA?

The lowest input requirement is 10 ng, but the more input you provide, the higher your sensitivity to detect low VAF.

18. Is the extraction technique important at this level of VAF?

Yes. The low concentration of ctDNA in non-advanced and non-metastatic cancer does limit the available concentration as a testing analyte to well below 10 ng/µL, so it is essential to have an efficient and scalable extraction technique to purify high-quality cfDNA samples.

19. Does this work for methylation detection?

No, this is for cfDNA variants and not to profile methylation status. We offer QIAseq Targeted Methyl Panels for detecting methylation status in your samples. You can learn more about these panels here.

20. What is the hands-on time?

About 2 hours.

21. For the booster panels, how many genes can be added?

You can add about 100 primers, so if your gene is short or if you’re looking at hotspots, you can add 8–10 genes. If you are looking for full coding regions, we recommend about 40 primers per gene, so you can add around 2 genes.

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