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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
[23, 24]
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
[28, 29]
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
[30, 31]
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
[32,33,34,35]
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
[43, 44]
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
[47, 48]
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
[50, 51]
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 [16, 46]
  1. Abbreviations: ctDNA circulating tumor DNA, PCR polymerase chain reaction, SNV single nucleotide variation, CNV copy number variation, SV structural variation, Bi-PAP bidirectional pyrophosphorolysis-activated polymerization, COLD Co-amplification at lower denaturation temperature, Tam-Seq Tagged-amplicon deep sequencing, Safe-SeqS Safe-Sequencing System, CAPP-Seq Cancer Personalized Profiling by deep sequencing, WES whole-exome sequencing, WGS whole- genome sequencing, SERS surface-enhanced Raman scattering, MALDI-TOF-MS matrix-assisted laser desorption/ ionization time of flight mass spectrometry, PARE personalized analysis of rearranged ends