Is there limitation of cfDNA amout in the NGS based gene testing?
Best insight from top research papers
The amount of cell-free DNA (cfDNA) available for Next Generation Sequencing (NGS) can be a limiting factor in gene testing. However, advancements in technology, such as Adaptor Template Oligo Mediated Sequencing (ATOM-Seq), have shown the ability to detect known mutations at very low allele frequencies using minimal DNA amounts. Additionally, the use of highly sensitive NGS technologies for analyzing cfDNA has emerged as a valuable alternative when tumor tissue is inadequate for biomarker analysis in lung cancer patients. Despite these advancements, challenges with distinguishing circulating tumor DNA from NGS artifacts persist, with early PCR errors being a principal source of noise in NGS data. Therefore, while limitations exist, innovative approaches and technologies are continuously improving the efficiency and accuracy of NGS-based gene testing using cfDNA.
Answers from top 4 papers
More filters
| Papers (4) | Insight |
|---|---|
| Yes, limitations exist due to errors in NGS of circulating cell-free DNA, impacting variant searches to known solid tumor mutations, necessitating sample duplicates for noise reduction and broader ctDNA applications. | |
| Yes, the paper highlights limitations of cfDNA amount in NGS-based gene testing for NSCLC, emphasizing the challenges and advantages of utilizing this approach for molecular profiling. | |
| Not addressed in the paper. | |
| ATOM-Seq technology allows detection of SNVs at 0.1% allele frequency using as little as 20 ng of cfDNA, demonstrating high sensitivity with minimal input material limitations in NGS-based gene testing. |
Related Questions
Is there an upper limit to the amount of cfDNA that can be tested in the NGS test?5 answersThe upper limit of cfDNA that can be tested in NGS tests depends on the specific assay used. Studies have shown that the OncomineTM Lung cell-free DNA Assay (OLcfA) demonstrated successful sequencing even with less than 10 ng of cfDNA, achieving a 0.1% limit of detection. Similarly, the UMIamp approach was able to detect mutations at levels as low as 0.002% in 60 ng cfDNA samples. Additionally, the Firefly assay showed a high detection rate of 98.89% at an allele frequency of 0.2% for 20 ng cfDNA. These findings suggest that while there may not be a strict upper limit, the sensitivity and efficiency of NGS assays can vary based on the amount of cfDNA tested, with some assays performing well even with minimal amounts.
How does the choice of ctDNA detection method - NGS versus PCR affect ctDNA detection?5 answersThe choice of ctDNA detection method, whether it is NGS or PCR, can affect ctDNA detection. NGS, or next-generation sequencing, is a multiplex sequencing platform that offers advantages such as higher sensitivity and the ability to detect multiple genomic alterations simultaneously. It has been shown to have high concordance rates with other methods like ddPCR. On the other hand, PCR, or polymerase chain reaction, is a traditional method that is less multiplexed and may have limitations in detecting certain alterations, especially in tumor-agnostic analysis. NGS-based ctDNA assays have been found to be highly consistent with ddPCR, indicating their value in detecting actionable mutations. Therefore, the choice of ctDNA detection method can impact the sensitivity, specificity, and multiplexing capabilities of the assay, ultimately affecting the detection of ctDNA alterations.
Issues with nanopore sequencing5 answersNanopore sequencing has several issues that need to be addressed. One challenge is the low base-call accuracy, which limits the reliability of the data for tasks such as antimicrobial resistance (AMR) and virulence gene detection, serotype prediction, and cluster identification. Another issue is the occurrence of incorrect and low confidence base calls in CNVs containing inverted duplicated sequences, which may be caused by secondary DNA structures impairing transit through the nanopore. Additionally, protein sequencing using nanopores remains highly challenging due to the greater complexity of the amino acid code and the variable charge across polypeptides. However, there have been advancements in nanopore technology, such as using 2-D materials like molybdenum disulfide (MoS2) nanopores for DNA and protein sequencing. Despite these challenges, nanopore sequencing has the potential for genomic epidemiology and public health investigations of bacterial pathogens, particularly in low-resource settings, due to its portability and affordability.
What is the limit of detection for macrolide resistance genes?4 answersThe limit of detection for macrolide resistance genes varies depending on the specific gene being detected. In one study, the lowest detection limit for the blaPSE gene was 6.6*10 copies/μL, while the lowest detection limit for the tetA, sul2, cmlA, qnrS, and aac(6')-Ib genes ranged from 24-202 copies/μL. Another study used a triplex PCR method to detect macrolide resistance genes mefA and ermB, and found that the presence of these genes accurately predicted macrolide resistance with erythromycin minimum inhibitory concentrations (MICs) ranging from 2-16 mg/L for strains with mefA and ≥256 mg/L for strains with ermB. The detection of mefA and/or ermB genes in non-culturable strains of pneumococci allowed for the prediction of erythromycin MICs with 93-100% precision. Overall, the limit of detection for macrolide resistance genes can vary depending on the specific gene and detection method used.
Does failure to extract a proper amount of dna can affect pcr test?3 answersFailure to extract a proper amount of DNA can indeed affect PCR tests. In one study, it was found that nucleotide substitutions in the human cytomegalovirus genome sequence led to decreased primer/probe binding, resulting in a 1,000-fold decrease in the sensitivity of the PCR assay. Another study focused on DNA extraction from highly processed samples and found that the method used significantly affected the yield and purity of the DNA extracts. Insufficient DNA extraction can lead to the generation of amplicons of less than 100 bp, which may not be suitable for PCR-based detection. Additionally, environmental samples often contain impurities that interfere with PCR amplification, and a rapid purification method was developed to obtain a DNA-containing extract pure enough for PCR amplification. Therefore, proper DNA extraction is crucial for the success and accuracy of PCR tests.
What are the limitations of challenge tests for genomic prediction?4 answersChallenge tests for genomic prediction have several limitations. Firstly, the evidence available to support genetic test use is often limited, which can hinder the implementation of new testing programs. Secondly, assessing the benefits and harms of genetic tests is challenging due to practical and ethical limitations of conducting meaningful clinical trials. Additionally, clinical endpoints may not capture the true benefit of genetic tests, as the main purpose of many tests is personal utility rather than medical benefit. Finally, the decision to have a predictive genetic test is influenced by psychological factors, and there is a lack of theoretical models to guide research in this area. These limitations highlight the need for further research and the development of appropriate assessment methods for genomic prediction tests.