Table 2.

Summary of commonly used molecular assays for the detection of ESR1 mutations.

Detection Method	Principle	Advantage	Limitation
NGS platforms	Massive parallel sequencing (mostly second generation)	Higher throughput and faster time than Sanger sequencing Detecting more dynamic range of genetic alterations than ddPCR	Time-consuming for data analysis Necessity of special knowledge for bioinformatics
Illumina	Amplification: Bridge PCR Sequencing: Synthesis using fluorescently labelled reversible terminator Detection: bases from individual images using camera	Higher throughput and accuracy than Ion Torrent because of the addition of a single base at a time reducing the homopolymer sequencing error	Partial overlap between emission spectra of the fluorophores and losing activity of fluorescent dyes limiting the base calling
Ion Torrent	Amplification: Emulsion PCR Sequencing: Semi-conductor sequencing utilizing hydrogen ion detection Detection: signals using ion sensor on the complementary metal-oxide-semiconductor chip	Longer reads and easier preparation and more direct, rapid, and less expensive than Illumina relying on laser scanners	Relatively low output and higher raw error rate due to vulnerability to insertions/deletions (indels) errors associated with homopolymeric stretches and repeats when compared to Illumina
ddPCR	Partitioning the reaction through water-oil emulsion droplets (emulsion-based digital PCR) or a chip with micro-channels (microfluidics-based digital PCR) Identified of target sequences using fluorescence and subsequent calculation the initial copy number and concentration of target molecules	Application to the detection of circulating tumor DNA based on high sensitivity with a detection limitation of 0.001% Absolute quantification via micro-partitioning Increase of the tolerance of PCR system to inhibitors because of the numerous micro-compartments Highly readable two-dimensional data Reliable results with small amount of samples because of the elimination error from pre-amplification High repeatability results based on its independence on amplification efficiency Lower cost compared to NGS platforms	Narrow dynamic range for genetic alterations compared to NGS technology Relatively higher cost than real-time quantitative PCR

NGS, next-generation sequencing; ddPCR, droplet digital polymerase chain reaction.