March 1, 2017 (Vol. 37, No. 5)
dPCR’s Partition-by-Partition Sequence-Counting Abilities Support Advances from Liquid Biopsies to Pathogen Detection
Digital PCR (dPCR) can accurately determine the copy numbers of targeted nucleic acid molecules in a sample, without requiring comparisons to reference standards or endogenous controls, provided that many reactions are performed in parallel. With this capability—along with others, including high measurement speeds and low operation costs—dPCR qualifies as a superior diagnostic tool for the clinic. In fact, dPCR is suitable for any application where extreme sensitivity or precise quantification is essential.
GEN recently spoke to several dPCR experts to get a sense of the current state of the field and where it might be headed.
GEN: What are the potential advantages and possible drawbacks of using dPCR over other methods when performing molecular measurements?
Dr. Hefner: The core of dPCR is partitioning. Partitioning has two key advantages. First, it greatly reduces the effects of PCR efficiency, obviating the need for standard curves and allowing for direct quantitation. Separating PCR efficiency from nucleic acid quantitation yields highly precise data output that drives elevated levels of reproducibility between users. Second, partitioning alleviates PCR product and resource competition within the reactions. This makes ultrasensitive detection and quantitation of highly related target sequences possible.
There are numerous published examples of mutant DNA being detected within a sea of wild-type sequences. One drawback for dPCR is that the workflow steps are often separate and require user interaction. However, new solutions that are on the horizon will likely streamline the dPCR process.
Mr. Apter: MilliporeSigma’s implementation of dPCR has improved and accelerated the custom cell engineering workflow. After zinc finger nuclease or CRISPR/Cas technology is applied to create precise genetic modifications in mammalian cell lines, dPCR is used to characterize the expected frequency of homologous recombination and develop a screening strategy. In some cell lines, homologous recombination occurs at a low frequency.
Digital PCR screens cell pools and identifies rare clones having the desired mutation. Digital PCR is also used to measure target gene copy number. The main disadvantage is that dPCR is reliant on the PCR amplification process. Therefore, dPCR is susceptible to the same difficulties that beset PCR. These include difficulties in getting good primer designs and amplifying regions characterized by GC-rich sequences, repetitive sequences, and secondary structures.
Ms. Venkatesh: The advantage of dPCR is the capability to count target molecules without standard curves or references. It provides a precise and accurate response even when detecting low signal (~0.1% limit of detection) in a high-noise background, and with minimal false positives. Digital PCR provides a linear response to allow small-fold differences to be detected, and it is less sensitive to PCR inhibitors.
These features make dPCR attractive for high-copy CNV detection, low-level pathogen detection, and liquid biopsy cancer applications. Although quantitative PCR (qPCR) is the gold standard, with its wide dynamic range, low cost per sample, and fast and simple workflows, it is limited by the need for comparison to a reference standard. Digital PCR and qPCR are complementary technologies, and together they enable a broad set of scientific applications.
GEN: What are some strategies and challenges for the accurate quantification of next-generation sequencing libraries with dPCR?
Dr. Hefner: A key challenge for accurate quantification is the dilution of the library material to fit within the dynamic range of the dPCR systems. Digital PCR platforms currently have a 3–5-log dynamic range depending on the system. This allows for detection of a couple molecules to ~100 K molecules/sample. Given that most libraries are highly concentrated, dilutions are required to allow for effective detection within the dPCR system.
Ms. Venkatesh: With the democratization of sequencing, the demand for lower cost of sequencing and higher quality of results from limited samples continues to rise. Conventional next-generation sequencing library quantification methods such as qPCR are challenged by insufficient sample quantity, and inefficient amplification of certain nucleotide sequences (AT- and GC-rich regions). This may prevent detection of rare variants and introduce bias.
Digital PCR provides a standard-curve-free method to precisely quantify next-generation sequencing libraries while minimizing sample handling. This provides a reliable and accurate library quantification strategy for efficient utilization of sequencer capacity and drives down the cost of sequencing.
GEN: For which particular clinical applications might dPCR be most appropriate?
Dr. Hefner: Digital PCR is often referred to as the gold standard for the detection of mutations in cell-free DNA. Numerous medical research institutes are devising digital PCR methods for monitoring for the presence of drug resistance markers in cancer. These studies are demonstrating earlier detection of disease recurrence. As this information becomes widespread, more and more physicians will likely start using residual disease monitoring as part of their treatment regimen for cancer patients.
Mr. Apter: Digital PCR is most advantageous for biopsies, early cancer detection, or any applications that rely on low-quality DNA templates or require the detection of low-abundance DNA or rare mutations. It is also appropriate for measuring fold-changes in expression.
Ms. Venkatesh: With its ability to sensitively and accurately detect mutant alleles in an abundance of wild-type signal, dPCR is gaining wider adoption in oncology laboratories, specifically in liquid biopsy tests, re-occurrence monitoring, and minimum residual disease detection. It is also used for orthogonal confirmation of results obtained using other techniques such as next-generation sequencing.
Additionally, dPCR applications in infectious disease are on the rise, as it can accurately determine pathogen loads at low levels and in the presence of PCR inhibitors such as hemoglobin.