TABLE I:

Current methods and challenges in cfDNA analysis

Challenge	Solution	Comments
DNA damage	DNA repair	Need high-yield method to reverse DNA oxidation and deamination
cfDNA sampling stochasticity	Larger blood volumes	Concern for patient health
cfDNA sampling stochasticity	Urine cfDNA	Need for extracting cfDNA from large volumes; process short cfDNA
Detecting mutations with ≤1% VAF	Digital PCR	Single-plex, only known mutations
Detecting mutations with ≤1% VAF	Mass spectrometry	Medium-plex, only known mutations
Detecting mutations with ≤1% VAF	NGS with UMIs	Expensive and low conversion yield
Detecting mutations with ≤1% VAF	NGS with allele enrichment	Low-plex, inaccurate quantitation, and low on-target rates
High conversion yield from cfDNA	N/A	Need high-yield end-repair and ligation
NGS depth uniformity	Primer/probe conc. tuning	labor-intensive and imperfect uniformity
Detecting fusions in cfDNA	Very large NGS panel	Very expensive because introns are long
Detecting CNVs in cfDNA	NGS, ddPCR	No current solution for detection ≤5% VAF
Rapid cfDNA diagnostics	Nanopore sequencing	High error rates and expensive reads
Affordable cfDNA diagnostics	N/A	Current cfDNA NGS panels have list price over $4,000