Direct quantification of high-load DNA samples with Countable PCR improves the sensitivity of rare event detection

Introduction

PCR is a key method for detection of rare biomarkers associated with disease [1]. While Rare Molecule Detection (RMD) is often considered in the context of minimal residual disease (MRD) for cancer, it is also used for early detection of infectious diseases such as viruses like HIV and HPV, as well as biodistribution studies in cell & gene therapy studies. RMD applications in the field are expanding, and researchers need genomic tools that push sensitivity limits, and enable broad, sensitive interrogation of biomarkers [2].

Presently, digital PCR (dPCR) is a common method for rare molecule detection, as it enables a relatively sensitive and scalable approach to estimate the Variable Allele Frequency (VAF), a common unit of measurement used in oncology. With dPCR, a %VAF limit of detection (LOD) of approximately 0.1% is considered sufficient for some use cases; but as precision and sensitivity demands increase, many seek to detect events at even lower frequencies. [3].

However, dPCR has notable drawbacks that limit its effectiveness as a method for rare molecule detection:

• dPCR uses Poisson statistics for quantification, which introduces estimation uncertainty.  Its limited partition capacity also caps the dynamic range even with Poisson statistics; detection saturates above about 5-logs, requiring dilution of high-abundance targets like wildtype alleles or housekeeping gens to avoid skewing %VAF.
• Additionally, microfluidics-based dPCR platforms suffer from low input capacity, reducing total analyzable DNA.  Achieving the sensitivity required for various applications often requires splitting samples across multiple reactions to overcome the low analyzable volume limitation of many dPCR-based systems.

With Countable PCR, we demonstrate the ability to enable detection of rare variants down to 0.004% VAF with minimal optimization and without special reagents. Here, we show an approach for expanding the search for rare molecules by loading a high amount of target DNA (3 μg). With Countable PCR’s broad dynamic range, it is straightforward to compare WT and mutant in the same reaction, in a single tube, without dilution or parallel reactions [4]. The result is very low VAF% sensitivity with good statistical confidence in a single reaction.

Materials

Reagents and consumables

Templates, Primers, and Probes

Note: “+” indicates locked nucleic acid (LNA) base. “/3IABkFQ/”  = 3′ Iowa Black quencher.

Experimental setup: Typical mass load for PCR

1. We prepared a V617F dilution series by spiking mutant DNA into wild-type gDNA, adjusting the WT mass so that total DNA per sample remained 100 ng. Four samples were prepared with 10-fold decreasing %VAF.
2. Each 100 ng spike-in sample was added to the Countable PCR amplification mix according to the Countable PCR Setup Guide [5].
3. Prior to matrix generation, we added EcoRI-HF (40 units) to each Countable PCR reaction and incubated the samples 37°C for 15 min to fragment the gDNA.
4. Continue the Countable PCR workflow and centrifuge the samples.
5. Prepared samples were then loaded into the Countable Instrument for imaging and automatic counting according to the Countable PCR Setup Guide.

Experimental setup: High mass load for PCR

1. We prepared a V617F dilution series by spiking mutant DNA into wild-type gDNA, adjusting the WT mass so that total DNA per sample remained 3000 ng. Five samples were prepared with 10-fold decreasing %VAF.
2. Each 3000 ng spike-in sample was added to the Countable PCR amplification mix according to the Countable PCR Setup Guide.
3. Prior to matrix generation, we added EcoRI-HF (40 units) to each Countable PCR reaction and incubated the samples 37°C for 15 min to fragment the gDNA.
4. Digest the samples at 37°C for 15 min to fragment the gDNA.
5. Continue the Countable PCR workflow and centrifuge the samples according to the Countable PCR Setup Guide.
6. Prepared samples were then loaded into the Countable Instrument for imaging and automatic counting according to the Countable PCR Setup Guide.

Counting and analysis

Counts were automatically produced in V 0.6 of The Countable Control software. To report results, the Countable Instrument directly images and counts fluorescent signal from amplified single-molecule targets and reports counts of target molecules per 50 μL reaction. Hundreds of light sheet images are taken, in less than 5 minutes per tube, and then processed automatically with the Countable Control Software (V0.6). Results are reported as direct counts; representative images are exported and displayed with results.

The LoD applied to find the threshold of reporting confidence was determined from the mean NTC signal (false-positive counts) plus two standard deviations from that background average.

Results

We compared the LOD for VAF% using a “typical” DNA mass load of about 100 ng versus a high input condition with about 3 μg of gDNA. Countable PCR’s matrix technology forms physically isolated compartments of single molecules, supporting unbiased amplification of multiple targets in a single tube. This enables detection across a broad dynamic range and minimizes the need to split DNA samples into multiple reactions to determine %VAF or reach the sensitivity required for some clinical applications.

Countable PCR outperforms off-the-shelf dPCR assays for “typical” DNA mass load of 100 ng

For typical input conditions, we began with approximately 100 ng DNA containing equal parts wild type gDNA and gDNA with JAK2 V617F mutation. Figure 1A shows representative images of dilution D3 from the JAK2 assay at 100ng input. Countable PCR processes light sheet images of the entire sample tube, maximizing analyzable volume. Figure 1B summarizes the results of the titration series, showing reliable detection down to 0.03% VAF.  Below 0.01% VAF, CVs become more variable.  Even with minimal assay optimization, Countable PCR outperformed typical off-the-shelf dPCR assays.

Figure 1: 100 ng JAK2 titration assay in Countable PCR.

(A) Representative images of WT and V617F fluorescent signals for approximately 100 ng load sample (Condition D2). Images were captured on the Countable system using light sheet imaging. Expected 10% VAF; Countable PCR measured 9% VAF.

(B) Full titration results with approximately 100 ng DNA input plotted. With “typical” mass load of DNA, Countable PCR measures to a limit of detection of about 0.03% VAF. Dilution 3 (Condition D3), expected to be 0.1% VAF, was well above the limit of detection (LoD); dilution 4 (Condition D4), with an expected VAF of 0.01% was below the LoD and therefore is not reported with confidence. For all conditions n = 4; LoD determination n = 16.

Countable PCR’s broad dynamic range and high sample processing volume enables 3 μg DNA load for vastly improved VAF% determination

To better leverage the high analyzable volume abilities of Countable PCR, we pushed the mass input to 3 μg of DNA in a one-tube reaction. The aim was to increase the number of rare events measured by the system by increasing the amount of input sample, without running more tubes. Though the maximum suggested DNA input for the Countable System is 1 μg, we loaded 3 μg to push the limits of the system. With 3 μg of input DNA, total counts (mutant + WT) for all tubes averaged 756789 – a number of targets that is too high for even high-sensitivity dPCR processing.

Figure 2A shows representative images of the typical load JAK2 VAF% assay for D4. Figure 2B shows the results of the 3 μg load JAK2 titration assay. The results show reliable detection down to 0.004% VAF, with CV becoming too variable below expected VAF of 0.001%. Here again, even with minimal optimization for the experiment, Countable PCR easily outperformed VAF% of typical dPCR assays, even those with special reagents and materials for boosting VAF% determination confidence.

Figure 2: 3 μg “high-load” JAK2 titration assay in Countable PCR.

(A) Representative images of WT and MUT for 3 μg load sample (from condition D3). Images were taken from a light sheet from the center-most part of the PCR tube during the scan. Expected VAF 0.1%; measured VAF 0.07%.

(B) Plotted results of JAK2 titration assay with 3 μg input DNA. For the high-mass load of DNA, Countable PCR measures to a limit of detection of about 0.004% VAF. Dilution 5 (condition D5), with an expected VAF of 0.005%, was in this case well above the LoB and therefore is reported as statistically significant. Dilution 3 (D3), dilution 4 (D4), and LoB n=2; dilution 5 (D5) n=3. LoD is pushed approximately ten-fold higher when using high-input samples.

Table 1 shows the summary of results from both experiments. Taken together, these results show that Countable PCR demonstrates superb out-of-the-box performance for rare molecule detection, pushing LOD to 0.004% in one tube per replicate without the need for extra experiments or special material processing.

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Table 1: Results from both typical load and high-load JAK2 DNA titration assays

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Conclusion

Countable PCR’s broad dynamic range makes it a practical and powerful tool for rare molecule detection. This is highly valuable for researchers who need to detect rare targets, as it allows them to broaden the search for rare molecules and achieve higher sensitivity detection, without extra experiments. With the ability to count rare and common events in the same tube, without concerns about Poisson saturation, Countable PCR delivers lower VAF detection with greater statistical confidence and less assay development.

As dPCR platforms approach fundamental limits for sensitivity, the demand for more advanced rare molecule detection methods continues to grow.  Countable PCR overcomes these limits by enabling highly sensitive and reliable detection of rare events in a streamlined workflow. With higher analyzable volume and a greater tolerance for high-frequency events, it reduces time, cost, and effort for rare molecule detection.

Resources

1. Qingqing Liu, Q. et al. Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review) Mol Med Rpt. 27(5) 1791-2997 https://doi.org/10.3892/mmr.2023.12991 (2023)
2. Pablo-Fontecha, V. et al. Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers. Sci Rep 13, 18997 https://doi.org/10.1038/s41598-023-39874-0 (2023)
3. Dai, P. et al. Calibration-free NGS quantitation of mutations below 0.01% VAF. Nat Commun.;12:6123. https://doi.org/10.1038/s41467-021-26308-6 (2021)
4. Liao, P. et al. Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion. PNAS. 117 (41) 25628-25633. https://doi.org/10.1073/pnas.2002448117 (2020)
5. Countable Labs. Countable PCR Setup Guide. www.countablelabs.com (2025)