Table 2 Summary of analytical approaches for ctDNA detection
From: Current status of ctDNA in precision oncology for hepatocellular carcinoma
Assay | Principle | Type of alteration | Limit of detection (mutant allele frequency) | DNA input | Evaluation | Reference | |
|---|---|---|---|---|---|---|---|
Real-time PCR | PCR primers with 3′ nucleotide extension utilizing mutated target genes | Known mutations | 10–20% | 2 ml of plasma | Easy to perform Qualitative analysis Unable to dynamic monitoring of cancer | ||
COLD-PCR | Utilizing the threshold temperature in the PCR, wild-type mutant heteroduplexes are selectively denatured to enrich for rare mutations | Known mutations | 0.01% | 25 pg-25 ng | Short time to output Enrich rare mutations Semi-quantitative | [25] | |
Bi-PAP | Primers with an overlapping nucleotide at the 3′ end activate the pyrophosphorolysis upon binding to the cognate template, thus allowing strand extension | Known mutations | 0.01% | 2 ml of plasma | Cost-effective Time-waste High error rate | [26] | |
Intplex | Mutant-specific primers are hybridized with a blocking oligonucleotide containing a phosphate group at the 3′ end to block the extension of the wild-type sequence | Known mutations | 0.004% | 2.25 pg/ml | Cost-effective Rapid data turnaround Pre-knowledge of genetic variants | ||
dPCR based | ddPCR | Involves millions of monodisperse droplets generated by microfluidic emulsification to create PCR microreactors that can perform millions of reactions in parallel | Known mutation | 0.001% | 5 ng/per reaction | Input amount depended sensitivity Easy to perform Pre-knowledge of genetic and epigenetic variants | |
BEAMing | Involves inputting pre-amplified products with primer-coated beads into limiting dilutions and performing further PCR reactions before the beads are purified and ligated to allele-specific fluorophore probes to distinguish between mutant and wild-type DNA | Known mutation | Less than 0.01% | 2 ml of plasma | High sensitivity Low sequencing cost Rapid when compared to NGS Pre-knowledge of genetic and epigenetic variants | ||
NGS based | TAm-Seq | Flexibly adapted to sequence multiple interested genomic regions in parallel by designing primers to amplify short amplicons | SNVs/indels/CNVs | 0.02% | 1 ml | Cost- and time effective High throughput Higher error rate | [37] |
Safe-SeqS | Tags each template DNA with unique molecular identifiers prior to amplification to create a unique family of sister molecules descended from the same original molecule | SNVs/indels | 0.1% | 3 ng | Improve the accuracy of massively parallel sequencing limited by the fidelity of the polymerase used in the PCR step | [38] | |
CAPP-Seq | Relied on a multiphase bioinformatics workflow to devise a “selector” for subsequent capture and sequence of mutated regions of interest | SNVs/indels/CNVs /Rearrangements | 0.02% | 32 ng | Low sequencing cost High coverage Improved Sensitivity Sequencing artifacts | [39] | |
Ion Torrent | Relies on standard DNA polymerase sequencing with unmodified dNTPs but uses semiconductor-based detection of hydrogen ions released during every cycle of DNA polymerization | SNVs/indels /CNVs/ fusions | 0.1% | 20 ng | Low sequencing cost High error rate | [40] | |
Methyl-Seq | Based on affinity, restriction enzyme or bisulfite conversion and utilize microarray or sequencing platforms downstream | Methylated regions | – | ~ 50 ng | Genome-wide coverage Bisulfite treatment damages the DNA | ||
WES | Amplification and sequence of the whole exome regions | SNVs/indels | More than 5–10% | 25 ng | Huge amounts of data per sample Low depth of coverage | [41] | |
WGS | Amplification and sequence of the whole genome regions | CNVs/SVs | – | 5-10 ng | High depth of coverage Costly | [42] | |
SERS | Multiplex mutation-specific primers amplify tumor DNA, followed by labeling of amplicons with specific SERS nanotags and enrichment with magnetic beads. Afterwards, Raman detection was performed to identify the mutations | SNVs | 0.1% | 2 ng/ul | Ultrasensitive Portable Bias in signal detection process Not yet applied in clinics | ||
MALDI-TOF-MS | Composed of multiplex PCR and mutation-specific single-base extension reactions while mutational genotypes are identified and characterized using matrix-assisted laser desorption/ionization time- of-flight mass spectrometry | SNVs | Less than 0.1% | ~ 10 ng | Multiple targets Ultrasensitive Unlimited sample throughput Few relevant studies on ctDNA | [49] | |
Electrochemical biosensor | The device incorporates immobilized DNA as a molecular recognition element on the electrode surface and with the introduction of nanostructured materials as interfacial film | SNVs | 0.01% | 12.5 k copies/μl or 20 ng in 10 μl | Time and cost-effective Rapid response Portability Not yet applied in clinics | ||
PARE | Biotin labels tag the ends of template sequences and then mate pairs are analyzed to identify intra-and inter-chromosomal rearrangements. | Genome-wide rearrangements | 0.001% | – | Whole genome coverage False-negative results | [46] | |
Digital karyotyping | Short genomic DNA tags were concatenated, cloned, and sequenced | chromosomally changed genomes/ new genomic regions | – | – | Rare clinical trials | ||