. Author manuscript; available in PMC: 2019 Jun 5.

Published in final edited form as: Pediatr Blood Cancer. 2019 Jan 6;66(5):e27595. doi: 10.1002/pbc.27595

TABLE 1. Features of PCR and NGS.

Listed are common features and limitations of PCR and NGS that should be considered when choosing a strategy for ctDNA assay development.

	PCR	Next Generation Sequencing
	PCR	Gene Panel¹	Translocation Panel¹	Shallow Sequencing (WIGS)	Deep Sequencing (WES/WGS)
Detectable variants	SNV/indel, translocation, CNA	SNV/indel, translocation, CNA	Translocation	CNA (Mb scale)	SNV/indel, CNA, Translocation (WGS)
Mutation-specific assay	Yes	No²	No³	No	No
Patient-specific assay	Yes (Translocation and non-recurrent variants)	No	No	No	No
Patient germline required	Variant dependent⁴	Variant dependent⁴	No	No	Yes
ctDNA quantification	ctDNA estimation complicated by CNA	Measuring multiple variants offsets effects of CNA	ctDNA estimation complicated by CNA	Multiple CNA improve ctDNA estimation	Measuring multiple variants offsets effects of CNA
Time to result	Day(s)	Week(s)	Week(s)	Week(s)	Week(s)
Relative cost	$	$$($)	$$	$$	$$$$
Sensitivity	Up to 0.005% of tumor fraction	Sensitivity increases with coverage⁵	Sensitivity increases with coverage	~3% (Adalsteinson et al.)	Sensitivity increases with coverage⁵
Detects novel variants	No	Novel variants in targeted gene(s)	Unknown translocation partners	Yes	Yes

^{1 -}

Gene and translocation panels can be combined because they use the same hybrid capture technology.

^{2 -}

Assays are not mutation specific but selected genes are typically restricted to those known to be mutated in the cohort of interest.

^{3 -}

Assays are not breakpoint specific but are enriched for intronic regions typically involved in the translocations of interest.

^{4 -}

For variants that can be observed in the germline (e.g. TP53), either germline control must be used or prior knowledge of germline status is required to interpret results.

^{5 -}

There is a risk of confusing sequencing artifact for low-allelic SNV/indels events. Error suppression strategies need to be incorporated when targeting these events by NGS.

SNV, single nucleotide variation; indel, small insertion or deletion; CNA, copy-number alterations; Mb, megabase.