Digital Assays Part I: Partitioning Statistics and Digital PCR

Abstract

A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, …). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error. The remainder focuses on digital PCR (dPCR) for quantification of nucleic acids. We discuss five commercial instruments that partition samples into physically isolated chambers (cdPCR) or droplet emulsions (ddPCR). We compare the strengths of dPCR (absolute quantitation, precision, and ability to detect rare or mutant targets) with those of its predecessor, quantitative real-time PCR (dynamic range, larger sample volumes, and throughput). Lastly, we describe several promising applications of dPCR, including copy number variation, quantitation of circulating tumor DNA and viral load, RNA/miRNA quantitation with reverse transcription dPCR, and library preparation for next-generation sequencing. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows. Part II focuses on digital protein and cell assays.

Formation of uniform reaction volumes using concentric amphiphilic microparticles

“…a microbead and a target cell or molecule inside a droplet approaches ~1% of the entire population of droplets, whereas the remaining droplets would have undesired combinations of the components. [4,14,15] Moreover, for multiplexing, multiple types of microbeads with distinct barcoding signatures should be encapsulated in separate droplets with the targets of interest, which is further limited by multiplicative probabilities to triple or larger Poisson distributions for duplex or greater multiplexing. Therefore, an instrument-free compartmentalization system that forms uniform droplets embedded with a single solid phase per droplet can address many of the challenges with current approaches.…”

Quantitation of Femtomolar‐Level Protein Biomarkers Using a Simple Microbubbling Digital Assay and Bright‐Field Smartphone Imaging

“…Although sensors in digital assays only need to distinguish between positive and negative signals, [10] digital ELISA mainly relies on fluorescent labels and requires asophisticated and nonportable laboratory-based high-resolution fluorescence microscopy system.…”

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples

“…There are two digital PCR partitioning methods that are chambers and droplets. Both methods have benefits, but the droplet digital PCR obtains a greater number of partitions that provide a more reliable quantification (Basu, ). The droplet digital PCR has been used for many animal and human viruses such as Japanese encephalitis virus (Wu et al., ), porcine reproductive and respiratory syndrome virus (Yang et al., ), orthopoxviruses (Americo, Earl, & Moss, ), Merkel cell polyomavirus (Arvia et al., ), infectious hematopoietic necrosis virus (Jia et al., ), human papilloma virus (Lillsunde Larsson & Helenius, ), human immunodeficiency virus (Dunay et al., ), hepatitis E virus (Nicot et al., ) and human cytomegalovirus (Sedlak, Cook, Cheng, Magaret, & Jerome, ).…”

“…There are two digital PCR partitioning methods that are chambers and droplets. Both methods have benefits, but the droplet digital PCR obtains a greater number of partitions that provide a more reliable quantification (Basu, 2017).…”