cfNOMe — A single assay for comprehensive epigenetic analyses of cell-free DNA

Erger, Florian; Nörling, Deborah; Borchert, Domenica; Leenen, Esther; Habbig, Sandra; Wiesener, Michael S.; Bartram, Malte P.; Wenzel, Andrea; Becker, Christian; Toliat, Mohammad Reza; Nürnberg, Peter; Beck, Bodo B.; Altmüller, Janine

doi:10.1186/s13073-020-00750-5

Cited by 48 publications

(54 citation statements)

References 38 publications

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“…In-house analysis of numerous cfDNA samples using TapeStation has shown that the majority of cfDNA fragments are indeed within the 50–200 bp range (with the median being ~167 bp, associating cfDNA with mononucleosomes [ 41 ]), showing, again, that contamination with germline DNA is minimalized (expected range for germline DNA is >10,000 bp). This has also been shown by others [ 42 , 43 , 44 ], confirming that robust pre-analytical handling of blood samples is a critical factor in cfDNA analysis [ 19 , 36 , 45 ]. Germline DNA was extracted from 200 μL of buffy coat using the QIAamp DNA Blood Mini kit (Qiagen), according to manufacturer’s instructions.…”

Section: Methodssupporting

confidence: 82%

Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression

Moss

Gorsia

Collins

et al. 2020

Cancers

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Despite the increasing incidence of endometrial cancer (EC) worldwide and the poor overall survival of patients who recur, no reliable biomarker exists for detecting and monitoring EC recurrence and progression during routine follow-up. Circulating tumor DNA (ctDNA) is a sensitive method for monitoring cancer activity and stratifying patients that are likely to respond to therapy. As a pilot study, we investigated the utility of ctDNA for detecting and monitoring EC recurrence and progression in 13 patients, using targeted next-generation sequencing (tNGS) and personalized ctDNA assays. Using tNGS, at least one somatic mutation at a variant allele frequency (VAF) > 20% was detected in 69% (9/13) of patient tumors. The four patients with no detectable tumor mutations at >20% VAF were whole exome sequenced, with all four harboring mutations in genes not analyzed by tNGS. Analysis of matched and longitudinal plasma DNA revealed earlier detection of EC recurrence and progression and dynamic kinetics of ctDNA levels reflecting treatment response. We also detected acquired high microsatellite instability (MSI-H) in ctDNA from one patient whose primary tumor was MSI stable. Our study suggests that ctDNA analysis could become a useful biomarker for early detection and monitoring of EC recurrence. However, further research is needed to confirm these findings and to explore their potential implications for patient management.

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Section: Methodssupporting

confidence: 82%

Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression

Moss

Gorsia

Collins

et al. 2020

Cancers

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“…TAPS and EM-Seq are sequencing methods based on enzymatic conversion of DNA (see previous section). EM-Seq has been used to develop cfNOMe (cell-free DNA-based nucleosome occupancy and methylation profiling), a single assay to evaluate the methylation status and nucleosome position on cfDNA from plasma and urine samples [ 63 ]. TAPS has also been successfully applied for cfDNA analysis [ 50 ].…”

Section: Experimental Assays For Dnam Evaluationmentioning

confidence: 99%

Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

et al. 2020

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Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.

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“…Lampignano et al suggested that downstream analytical processes, such as massively parallel sequencing (MPS) and droplet digital PCR, could be utilized with a variety of collection tubes [ 161 ], however, there are some exceptions, usually when methylation analyzes should be performed. For example the Streck Cell-Free DNA BCT tubes do not seem to work for such analyzes, therefore the use of the Qiagen PAXgene Blood ccfDNA Tubes should be considered when designing methylation assays [ 106 ], however, there are also reports of the use of the Streck Cell-Free DNA BCT tubes for epigenetic analysis of cfDNA [ 162 ]. Although K 2 EDTA showed worse effect then the Streck Cell-Free DNA BCT tubes on the cfDNA and ctDNA recovery, it seems that on the quality of MPS analyzes neither of these preservatives have noticeable effect [ 26 ], even the Streck Cell-Free DNA BCT tubes can be used for the library preparation for aneuploidy detection in prenatal care without any unwanted changes [ 93 ].…”

Section: Sample Collectionmentioning

confidence: 99%

“…Using a microarray technology approximately 3000 different mRNAs were discovered in cell-free saliva. It points to that not only from blood, but cfNAs originating from all body fluids should be acquired in sufficient quality and quantity for plenty of downstream analysis regardless of whether they are performed in a diagnostic manner or in research [ 162 , 163 ].…”

Section: Sample Collectionmentioning

confidence: 99%

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

Pös

Styk

et al. 2020

IJMS

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Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.

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cfNOMe — A single assay for comprehensive epigenetic analyses of cell-free DNA

Cited by 48 publications

References 38 publications

Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression

Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression

Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

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