cfDNA Sequencing: Technological Approaches and Bioinformatic Issues
Abstract
:1. Introduction
2. Pre-Analytical Requirements
3. Detection of ctDNA by Sequencing Technologies
3.1. PCR-Based Methods
3.1.1. Quantitative PCR
3.1.2. Digital PCR
3.1.3. PCR Coupled with Mass Spectrometry
3.2. Targeted NGS-Based Methods
- Tagged-amplicon deep sequencing (Tam-Seq)
- Safe-Sequencing System (Safe-SeqS)
- Duplex sequencing
- Targeted error correction sequencing (TEC-Seq)
- Single primer extension (SPE) with unique molecular barcode
- Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq)
- Immunoglobulin high-throughput sequencing (Ig-HTS)
3.3. Untargeted NGS-Based Methods
4. Bioinformatical Methods
4.1. Adapter Contamination
4.2. Library Biases and Molecular Barcoding
4.3. Bioinformatics Processing
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Analysis Type | Technique | Sensitivity (LoD) | Targets | Applications | Advantages | Limitations | |
---|---|---|---|---|---|---|---|
PCR based methods | qPCR | ARMS-PCR | 0.01–0.1% | Hotspot mutation | Cancer detection and monitoring, targetable alterations, some assays approved for clinical use | High specificity and sensitivity, cost effective, rapid, ease of use | No multiplexing, limited to detection of known mutations |
PNA-LNA Clamp PCR | |||||||
COLD PCR | |||||||
digital PCR | ddPCR | 0.01–0.1% | Hotspot mutations, gene fusions, CNV | Cancer detection and monitoring, targetable alterations, some assays approved for clinical use | Up to 5 targets, high sensitivity and specificity, absolute quantification, single molecule analysis, cost effective, rapid, ease of use | Limited multiplexing (number of fluorescent colors), limited to detection of known mutations | |
BEAMing | |||||||
PCR coupled to spectrometry | SERS | 0.1–1% | Known mutations | Cancer detection and monitoring, targetable alterations, for research use | Multiplexing capacity | Limited to detection of known mutations | |
PCR based methods | UltraSEEK | ||||||
NGS based methods | targeted | Tam-Seq | 2% | Known and unknown mutations, indels, CNV, chromosomal rearrangements (capture) | Cancer detection and monitoring, classification, targetable alterations, for research use | High specificity | Amplicon methods by multiplex PCR (depend on fragment size), no error correction |
eTam-Seq | 0.02% | Error correction | Amplicon methods by multiplex PCR | ||||
Safe-SeqS | 0.01–0.05% | Error correction by SSCS | Amplicon methods by multiplex PCR | ||||
Duplex sequencing | 0.0001–0.1% | Error correction by DSCS | Amplicon methods by multiplex PCR | ||||
TEC-Seq | 0.05–0.1% | Error correction by SSCS, Hybrid capture method (not dependent on fragment size) | Less comprehensive than WGS or WES | ||||
single primer extension (SPE) | 0.5–1% | Amplicon methods by SPE (not dependent on fragment size), error correction by SSCS | Less comprehensive than WGS or WES | ||||
SPE-duplex UMI | 0.1–0.2% | Error correction by DSCS | Less comprehensive than WGS or WES | ||||
CAPP-Seq | 0.02% | Hybrid capture method (not dependent on fragment size) | Need large input, allelic bias (capture), stereotypical errors (hybridization step), less comprehensive than WGS or WES | ||||
iDES eCAPP-Seq | 0.00025–0.004% | Error correction by DSCS and correction of stereotypical errors | Less comprehensive than WGS or WES | ||||
Ig-HTS | 0.001% | VDJ rearrangements | Non-invasive monitoring, approved for clinical use | Very high sensitivity | Tissue biopsy needed | ||
Untargeted | WES | 5% | Coding regions, intron-exon junctions, promoters, untranslated regions, non-coding DNA of miRNA genes | Cancer detection, monitoring of resistant clones in metastasis, for research use | Mutation discovery and signatures, detection of CNV, fusion genes, rearrangements, predicted neoantigens and Tumor Mutational Burden | Low sensitivity (increasing depth lead to high cost), need bioinformatic expertise | |
WGS | 5–10% | Structural variants (fragmentation pattern, genome-wide CNV, methylation profile) | Cancer localization and origin, early detection (early and late stage), for research use | Shallow sequencing, genome wide profiling, identification of cancer signatures | Expensive, variable sensitivity (low) and specificity, need bioinformatic expertise, lots of data generated |
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Bohers, E.; Viailly, P.-J.; Jardin, F. cfDNA Sequencing: Technological Approaches and Bioinformatic Issues. Pharmaceuticals 2021, 14, 596. https://doi.org/10.3390/ph14060596
Bohers E, Viailly P-J, Jardin F. cfDNA Sequencing: Technological Approaches and Bioinformatic Issues. Pharmaceuticals. 2021; 14(6):596. https://doi.org/10.3390/ph14060596
Chicago/Turabian StyleBohers, Elodie, Pierre-Julien Viailly, and Fabrice Jardin. 2021. "cfDNA Sequencing: Technological Approaches and Bioinformatic Issues" Pharmaceuticals 14, no. 6: 596. https://doi.org/10.3390/ph14060596
APA StyleBohers, E., Viailly, P. -J., & Jardin, F. (2021). cfDNA Sequencing: Technological Approaches and Bioinformatic Issues. Pharmaceuticals, 14(6), 596. https://doi.org/10.3390/ph14060596