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 | Â | [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. | Â | [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. | |
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] |