cfDNA Sequencing: Technological Approaches and Bioinformatic Issues

Bohers, Élodie; Viailly, Pierre‐Julien; Jardin, Fabrice

doi:10.3390/ph14060596

Cited by 39 publications

(33 citation statements)

References 134 publications

(148 reference statements)

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“…Over the last several years, significant progress has been made in the development of ctDNA detection methods. PCR-based sequencing, which includes real-time quantitative PCR (qPCR), and digital PCR (dPCR) methods, is an alternative method for single-locus/multiplexed tests and targeted panels, while Next Generation Sequencing (NGS)-based sequencing, which includes Tagged-Amplicon deep sequencing (TAM-Seq), CAncer Personalized Profiling by deep sequencing (CAPP-Seq), and Duplex sequencing can be applied to panels of any size ( 1 , 2 ). Notably, the revolution in ctDNA-based liquid biopsies has opened up new opportunities for cancer diagnosis, prognosis, monitoring, and treatment guidance ( 3 ).…”

Section: Introductionmentioning

confidence: 99%

Circulating Tumor DNA and Minimal Residual Disease (MRD) in Solid Tumors: Current Horizons and Future Perspectives

Peng

Mei

et al. 2021

Front. Oncol.

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Circulating tumor DNA (ctDNA) is cell-free DNA (cfDNA) fragment in the bloodstream that originates from malignant tumors or circulating tumor cells. Recently, ctDNA has emerged as a promising non-invasive biomarker in clinical oncology. Analysis of ctDNA opens up new avenues for individualized cancer diagnosis and therapy in various types of tumors. Evidence suggests that minimum residual disease (MRD) is closely associated with disease recurrence, thus identifying specific genetic and molecular alterations as novel MRD detection targets using ctDNA has been a research focus. MRD is considered a promising prognostic marker to identify individuals at increased risk of recurrence and who may benefit from treatment. This review summarizes the current knowledge of ctDNA and MRD in solid tumors, focusing on the potential clinical applications and challenges. We describe the current state of ctDNA detection methods and the milestones of ctDNA development and discuss how ctDNA analysis may be an alternative for tissue biopsy. Additionally, we evaluate the clinical utility of ctDNA analysis in solid tumors, such as recurrence risk assessment, monitoring response, and resistance mechanism analysis. MRD detection aids in assessing treatment response, patient prognosis, and risk of recurrence. Moreover, this review highlights current advancements in utilizing ctDNA to monitor the MRD of solid tumors such as lung cancer, breast cancer, and colon cancer. Overall, the clinical application of ctDNA-based MRD detection can assist clinical decision-making and improve patient outcomes in malignant tumors.

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Section: Introductionmentioning

confidence: 99%

Circulating Tumor DNA and Minimal Residual Disease (MRD) in Solid Tumors: Current Horizons and Future Perspectives

Peng

Mei

et al. 2021

Front. Oncol.

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“…Similarly, the need to detect low mutation rates within the drug-resistant Mycobacterium tuberculosis strains 19 or drug-resistance colorectal cancer 20 has been reported. Some common qPCR diagnostics methods using DNA polymerases lacking proofreading activity, like blocker displacement amplification (BDA) 21 , allele-specific PCR (As-PCR) 22,23 , amplification-refractory mutation system (ARMS-PCR) 24,25 , competitive allele-specific TaqMan PCR (Cast-PCR) 26,27 , and coamplification at lower denaturation temperature PCR (COLD-PCR) 28 , mainly have a limit of detection (LoD) around 0.1% to 1% VAF which is no longer satisfactory 29 . The high polymerase misincorporation rate 30 from the lack of the proofreading activity could introduce false-positive mutations into generated amplicons, thus obscuring readouts in qPCR methods and restricting the LoD (Figure S1A).…”

Section: Introductionmentioning

confidence: 99%

Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Zhang

Pinto²,

Dai

et al. 2021

Preprint

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Effective polymerase chain reactions (PCR) are important in bio-laboratories. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. High-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. Yet, there are currently no proper designs for multiplexed HiFi qPCR reactions. Moreover, the popularity of targeting highly multiplex DNA sequences requires minimizing PCR side products, as the potential of dimerization grows quadratically as the plexes of primers increases. Efforts tried before were either an add-on step, or technology-specific, or requiring high-level computing skills. There lacks an easy-to-apply and cost-effective method for dimerization reduction. Here, we presented the Occlusion System, composed of a 5’-overhanged primer and a probe with a short-stem hairpin. We demonstrated that it allowed multiplexing high-fidelity qPCR reaction, it was also compatible with the current variant-enrichment method to improve the LoD by 10-fold. Further, we found that the Occlusion System reduced the dimerization up to 10-fold in highly multiplexed PCR. Thus, the Occlusion System satisfactorily improved both qPCR sensitivity and PCR efficiency.

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“…Liquid biopsy failed to detect tumor-specific variants that were presented in the tumor with low VAF, which strongly suggests that more sensitive sequencing approaches are needed to capture these variants reliably. In practice, this often means even deeper sequencing or the use of more sophisticated methods such as unique molecular identifiers [ 43 ]. At the same, there is an apparent pressure to perform liquid biopsy as cost-effectively as possible so that the liquid biopsy can truly compete with tumor DNA sequencing which is often more affordable option in clinics.…”

Section: Discussionmentioning

confidence: 99%

Circulating Cell-Free DNA Reflects the Clonal Evolution of Breast Cancer Tumors

Kujala

Hartikainen

Tengström

et al. 2022

Cancers

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Liquid biopsy of cell-free DNA (cfDNA) is proposed as a potential method for the early detection of breast cancer (BC) metastases and following the clonal evolution of BC. Though the use of liquid biopsy is a widely discussed topic in the field, only a few studies have demonstrated such usage so far. We sequenced the DNA of matched primary tumor and metastatic sites together with the matched cfDNA samples from 18 Eastern Finnish BC patients and investigated how well cfDNA reflected the clonal evolution of BC interpreted from tumor DNA. On average, liquid biopsy detected 56.2 ± 7.2% of the somatic variants that were present either in the matched primary tumor or metastatic sites. Despite the high discordance observed between matched samples, liquid biopsy was found to reflect the clonal evolution of BC and identify novel driver variants and therapeutic targets absent from the tumor DNA. Tumor-specific somatic variants were detected in cfDNA at the time of diagnosis and 8.4 ± 2.4 months prior to detection of locoregional recurrence or distant metastases. Our results demonstrate that the sequencing of cfDNA may be used for the early detection of locoregional and distant BC metastases. Observed discordance between tumor DNA sequencing and liquid biopsy supports the parallel sequencing of cfDNA and tumor DNA to yield the most comprehensive overview for the genetic landscape of BC.

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cfDNA Sequencing: Technological Approaches and Bioinformatic Issues

Cited by 39 publications

References 134 publications

Circulating Tumor DNA and Minimal Residual Disease (MRD) in Solid Tumors: Current Horizons and Future Perspectives

Circulating Tumor DNA and Minimal Residual Disease (MRD) in Solid Tumors: Current Horizons and Future Perspectives

Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Circulating Cell-Free DNA Reflects the Clonal Evolution of Breast Cancer Tumors

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