Biology subjects

Van Paemel, R.

Publications and source records attributed to Van Paemel, R..

7 recordsLinked to original sources

bioRxiv2025

A benchmark of DNA methylation deconvolution methods for tumoral fraction estimation using DecoNFlow

In cancer patients, circulating cell-free DNA (cfDNA) is released into body fluids from both healthy and cancer cells. The proportion of tumor-derived cfDNA serves as a surrogate marker of tumor burden allowing disease monitoring. Tumoral cfDNA can be distinguished based on patient specific tumoral mutations or using more general tumor specific DNA methylation patterns, that are preserved on tumoral cfDNA. DNAm profiling of cfDNA thus enables non-invasive cancer detection and monitoring. However, accurately determining tumour fractions remains challenging due to the heterogeneous mixture of cfDNA sources in body fluids. Computational DNAm deconvolution methods address this by inferring cell-type contributions either with or without reference methylomes. While several tools exist and multiple benchmarking studies have been performed, none have specifically evaluated the sensitivity and accuracy of tumour-fraction estimation in cfDNA-focused contexts. Here, we benchmarked 10 reference-based and 2 reference-free DNAm deconvolution tools using 3,690 in silico mixtures spanning multiple tumour types, different bisulfite-based sequencing strategies and several sequencing depths. Overall, CelFiE showed the most accurate tumour-fraction estimation across the different conditions. Interestingly, reference-free methods demonstrated superior sensitivity for tumour detection, but consistent over-estimation of tumoral fraction. We further observed that sequencing depth strongly affects performance until sufficient saturation is achieved. To enable reproducible evaluation and tool selection within this benchmark, we developed DecoNFlow, a scalable Nextflow pipeline integrating 12 deconvolution tools and 3 marker selection methods, making it the most comprehensive pipeline for sequencing-based deconvolution up to date. Together, our findings provide practical guidance for tool selection in cfDNA tumour monitoring and establish DecoNFlow as a robust framework for benchmarking and applying DNAm deconvolution.

bioinformatics↗

bioRxiv2021

Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

The use of blood-based extracellular RNA (cell-free RNA; exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing, and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated ten blood collection tubes, three time intervals between blood draw and downstream processing, and eight RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics on deep transcriptome profiling of both small and messenger RNA from healthy donors plasma or serum was assessed for each pre-analytical variable separately and for interactions between a selected set of pre-analytics, resulting in 456 extracellular transcriptomes. Making use of 189 synthetic spike-in RNAs, the processing and analysis workflow was controlled. When comparing blood collection tubes, so-called preservation tubes do not stabilize exRNA well, and result in variable RNA concentration and sensitivity (i.e., the number of detected RNAs) over time, as well as compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity, and demonstrate interactions between specific blood collection tubes, purification kits and time intervals. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for a given experiment. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs), representing paramount groundwork for future exRNA-based precision medicine applications.

genomics↗

bioRxiv2020

When DNA gets in the way in RNA-seq experiments, a sequel

Using a newly developed method dubbed SILVER-Seq--enabling extracellular RNA sequencing (exRNA-seq) directly from a small volume of human serum or plasma-- Yan et al. recently reported in Current Biology a potential exRNA biomarker for the early diagnosis of Alzheimers disease [1]. After the publication of the initial paper describing the SILVER-Seq method [2], we reported our concern regarding potential DNA contamination in their datasets [3]. Although the authors replied they were able to successfully treat RNA samples with DNase to avoid such contamination, they did not address our observations of the majority of reads without evidence of being derived from RNA, nor documented verified absence of DNA after DNase treatment [4]. To assess whether the newly data generated may suffer from DNA contamination, we downloaded the publicly available sequencing data and evaluated two quality control metrics (i.e., fraction of exonic and splice reads), which were not reported in the paper. We found that both quality metrics were much lower than expected for RNA-seq data (6.28% exonic and 0.478% splice reads), in line with our previous findings on the first SILVER-Seq paper. These observations suggest the data and results presented by Yan et al. are affected by DNA contamination, an issue that may be inherent to the SILVER-Seq technology.

molecular biology↗

bioRxiv2020

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

BackgroundThe methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulfite-based cfDNA methylation profiling. Methods15 tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulfite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. ResultsAll preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. ConclusionThe methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

genomics↗

bioRxiv2019

Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

In the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

genomics↗

bioRxiv2019

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

molecular biology↗

bioRxiv2019

A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

Methylation profiling of circulating cell-free DNA (cfDNA) is of great interest as a liquid biopsy assay for the detection and monitoring of cancer and other pathologies. Here we describe circulating cell-free DNA reduced representation bisulfite sequencing (cf-RRBS), enabling the use of highly effective RRBS on fragmented plasma cfDNA. This method enriches the CpG-rich RRBS target regions by enzymatic degradation of all off-target DNA rather than by targeted capture, in contrast to previous methods. Critical steps are fully enzymatic in a single-tube, making it rapid, cost-effective, robust, and easily implemented on a liquid-handling station for high-throughput sample preparation. We benchmark cf-RRBS results to those obtained by previous more complex methods and exemplify its use for accurate non-invasive subtyping of lung cancer, a frequent onco-pathology task. cf-RRBS enables any molecular pathology lab to tap into the cfDNA methylome, only making use of off-the-shelf reagents and open-source data analysis tools. One Sentence SummaryNovel methodology enables facile and cost-effective methylation profiling of fragmented plasma DNA, allowing for routine liquid biopsy clinical diagnostics, exemplified here for differential diagnosis of lung cancer subtypes.

genomics↗