Table 2

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

Droplet digital PCRWhole exome next generation sequencing (NGS)Targeted NGSTargeted NGS with molecular barcodesTargeted NGS with molecular inversion probes (MIPs)
PrinciplePartitioning of single DNA templates into micro-droplets, followed by end-point PCRs and enumeration of fluorescent dropletsHigh-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 NGSMIPs, which carry tag sequences, are used to capture targeted DNA regions via hybridization prior to sequencing
Comprehensiveness of testing; potential clinical/research usesLow; suitable for small numbers of “hotspot” mutationsHigh; suitable for defining reference mutational landscapes of cancersModerate to high, depending on the panel; suitable for clinical testing or biomarker research once the recurrently mutated genes are known
Barcoding/error correctionN/ANoYes
Typical limit of detection0.01%–0.1%As low as 2%–3% <0.1%
Amount of input DNA requiredLowMedium to highMedium
Samples per runVariable, depending on number of tests and platformRelatively few samplesMore samples than WES
Other consideration/ commentsAbsolute quantification of VAFsVAFs 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.