Table 2

Molecular technologies used for the assessment of CH: a comparison of approaches

	Droplet digital PCR	Whole exome next generation sequencing (NGS)	Targeted NGS	Targeted NGS with molecular barcodes	Targeted NGS with molecular inversion probes (MIPs)
Principle	Partitioning of single DNA templates into micro-droplets, followed by end-point PCRs and enumeration of fluorescent droplets	High-throughput sequencing of all coding regions from the human genome (ie, the exome)	High-throughput sequencing of selected exons and genes recurrently mutated in cancers (eg, myeloid neoplasms)	Short molecular barcode sequences are attached to DNA fragments during library construction; otherwise, similar to targeted NGS	MIPs, which carry tag sequences, are used to capture targeted DNA regions via hybridization prior to sequencing
Comprehensiveness of testing; potential clinical/research uses	Low; suitable for small numbers of “hotspot” mutations	High; suitable for defining reference mutational landscapes of cancers	Moderate to high, depending on the panel; suitable for clinical testing or biomarker research once the recurrently mutated genes are known
Barcoding/error correction	N/A	No		Yes
Typical limit of detection	0.01%–0.1%	As low as 2%–3%		<0.1%
Amount of input DNA required	Low	Medium to high		Medium
Samples per run	Variable, depending on number of tests and platform	Relatively few samples	More samples than WES
Other consideration/ comments	Absolute quantification of VAFs	VAFs are approximate		Near absolute quantification of VAFs Additional cost of molecular barcodes in comparison to targeted NGS without barcodes	Near absolute quantification of VAFs Near absolute quantification of VAFs

PCR-based testing, while analytically sensitive and inexpensive, is often restricted to the assessment of small numbers of ‘hotspot’ mutations. By contrast, sequencing in general allows for the more comprehensive evaluation of larger complements of mutations. Among sequencing approaches, WES, while comprehensive, is comparatively limited in terms of depths of coverage (limited analytical sensitivity) and the numbers of samples that can be sequenced at one time. The incorporation of molecular barcodes and similar tag sequences in some workflows allows for the distinction between low-level mutations and sequencing artefacts, thereby greatly improving the analytical sensitivity for low-level mutations.
MIPs, molecular inversion probes; NGS, next-generation sequencing; VAFs, variant allele fractions; WES, whole exome sequencing.