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.

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