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Table 1 Common ctDNA Analysis Techniques. Description of Twelve Common Methodologies of ctDNA Analysis

From: The interplay of circulating tumor DNA and chromatin modification, therapeutic resistance, and metastasis

Technique

Main features

Description

Accuracy, sensitivity, specificity

Advantages

Challenges or perspectives

Ref.

Hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq)

A hybridization-based method sequencing all protein-coding exons

Barcoded cfDNA-seq libraries design, probe hybridization, target capture, post-capture amplification and bead clean up of captured amplified DNA

AFs: 0.25%

specificity: 98%

1. High fidelity

2. Screening for mutations throughout a diversity of genomic regions

Larger portions of the genome to query other target genes or mutation classes like rearrangements and copy number alterations

[39]

DNA clutch probes (DCP)

Without enzymatic amplification but a DCP used to prevents the reassociation of ssDNAs

ctDNA denaturization, DCP preventing reassociation of ssDNA, PNA clamps hybridizing to the matched wild type, detection of remaining single-stranded mutant target ctDNA

Detect 0.01% mutations

1. High specificity with less time requirement

2. Chip-based format supports automation.

Monitoring diseases caused by DNA viruses

[40]

iDES-enhanced CAPP-Seq

Combining in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules

Designing ‘index’ barcode and‘insert’ barcodes, PCR, mapping to reference genome to recover single strand, duplex recovery, in silico reassembly of original DNA duplex

4 in 105 cfDNA molecules

Increased scalability, flexibility, coverage uniformity, and ability to reliably assess all mutation classes in a single assay

Allowing for greater analytical sensitivity than iDES if >~200 somatic mutations were targeted

[41]

Targeted error correction sequencing (TEC-Seq)

A direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing

including dual-index barcode adapters design, cfDNA library formation, redundant sequencing of the library, reconciliation of duplicate fragments, alignment to the reference genome, identification of bona fide alterations.

Sensitivity: 97.4%

specificity>99.9999%

Sensitive and highly specific detection of low-abundance sequence alterations using NGS

Sensitivity may be further improved by deeper sequencing, improved error correction methods, larger blood volumes, and repeated testing at regular intervals.

[19]

Nanoplasmonic biosensor

Localized surface plasmon resonance (LSPR) and the coupling plasmon mode of gold nanoparticles (AuNPs) for enrichment strategy.

A change of the refractive index surrounding the biosensor surface for binding of ctDNA to the PNA-probed AuNP surface. Change of RI as distinct LSPR-peak changes on the Rayleigh light scattering. Detection and amplification of methylation by specifically binding immunogold colloids

Sensitivity: four times (~50 fM) improvement

Simultaneous detection of the hot-spot mutation and epigenetic changes

on the ctDNA

Providing sharp sensitive and multiplexed platform for detecting other associated biomarkers and their modifications at low concentration.

[42]

Simple multiplexed PCR-based barcoding

of DNA

Detection of extremely rare variant alleles within a complex mixture of DNA molecules

Comprising a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing

Error correction to <0.1%,

1.Simplicity of the NGS

library construction protocol and the ease in any reasonably capable research laboratory

2. The low DNA input (<5 ng),

1. time-consuming, and not be the best approach for coverage of consistent, large target regions on many samples.

2. Requirement of deep sequencing, and sequencing costs

[43]

Sensitive digital quantification of DNA methylation in clinical samples

Providing an opportunity to assess DNA methylation with allele-specific PCR, restriction digestion or specific hybridization probes

Digital approaches involve the counting of methylated and unmethylated fragments, one-by-one, thereby dramatically increasing the signalto-noise ratio of the assay.

the methylated DNA fraction was 0.018%

1.enabling increased sensitivity and specificity 2.enabling comparisons across different patient cohorts for standardized clinical interpretations

 

[44]

Nanostructured conductive polymer platform

Extracting tumor-specific circulating cfDNA from unprocessed plasma using an electroactive Ppy/Au NW platform

Ppy-coated Au nanowires (Ppy/Au NWs) capture DNA with oxidation electric fields by DNA-Ppy surface adsorption, while Ppy/Au NWs release DNA with reduction electric fields.

mean purity: 1.97 ± 0.02

Enhanced efficiency, high yield and high purity

_

[45]

Tagged-amplicon deep sequencing (TAm-Seq)

Combining short amplicons, two-step amplification, sample barcodes with high-throughput PCR

Preamplification of DNA molecules with or without mutations, single-plex PCR to select region of interest, barcoding PCR to harvest amplicons duplicate sequencing to avoid false positives caused by PCR errors

AF: 2%

1. A balance between sensitivity and ease of use

2. Effective amplification

3. Sample barcodes and high-throughput PCR

4. less time

Challenge: detection limit compared to assays that target individual loci

[46]

Single copy sensitive electrochemical Assay

Schematic representation of the SEDA strategy.

Integrated by dual sequence discrimination processes including methylation-specific annealing and specific interface hybridization, as well as cascade signal amplification processes represented by the asymmetric MSP and HRP catalytic reaction.

The high specificity reaching a 0.1% methylation index

1. Integrated by dual sequence discrimination processes and cascade signalamplification processes

2. Detection of tumor related methylation in lung cancer patients with 200 microlitre plasma samples.

 

[48]

Improved hMe-Seal

Determining the genome-wide distribution of 5-hmC by selective labeling as enrichment strategy

Using the T4 bacteriophage beta-glucosyltransferase to install a glucose moiety with an azide group onto the hydroxyl group of 5-hmC. then labeled with biotin, thus enables 5-hmC–containing DNA detection, capture, enrichment and sequencing

Detection limit: ~0.004%

Providing acurate and comprehensive capture of 5-hmC–containing DNA fragments, while still providing high selectivity.

Enable us to understand the role(s) of the 5-hmC modification at molecular, cellular and physiological levels.

[49, 50]

Discrimination of Rare EpiAlleles by Melt ( DREAMing )

Semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species

cfDNA extraction, bisulite conversion, sample dilution, PCR amplification and derivative melt profile analysis. Melt profile shows a secondary melt peak for fully methylated and heterogeneously-methylated epiallele while melt curve of the unmethylated presents only one peak.

Single-CpG-site resolution in fractions: 0.005%

1.Minimal time and cost using a standard qPCR machine and microtiter plate.

2.‘DREAM analysis’ histogram helps easily visualize epigenetic/epiallelic heterogeneity.

1. The sensitivity of the assay determined by the dominant epiallelic methylation density, and epiallelic species not be accurately represented.

2. Relatively low throughput.

3. Not directly provide sequence information.

[51]

  1. Description of Twelve Common Methodologies of ctDNA Analysis. These developed assays and protocols enable excellent accuracy, sensitivity and specificity in the detection of ctDNA and its variation. And those approaches have their own advantages and perspectives. Those approaches include hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq), DNA clutch probes (DCP), integrated digital error suppression (iDES)-enhanced CAPP-Seq, targeted error correction sequencing (TEC-Seq), nanoplasmic biosensor, Simple multiplexed PCR-based barcoding of DNA, Sensitive digital quantification of DNA methylation, Nanostructured conductive polymer platform, tagged-amplicon deep sequencing (TAm-Seq), Single copy sensitive electrochemical assay, Improved hMe-Seal and Discrimination of Rare EpiAlleles by Melt (DREAMing). (AF: allele frequencies)