Biology subjects

Pegtel, D. M.

Publications and source records attributed to Pegtel, D. M..

3 recordsLinked to original sources

bioRxiv2022

Circulating extracellular vesicles in lung cancer patients are not enriched in tumor-derived DNA fragments as revealed by whole genome sequencing.

Liquid biopsies contain multiple analytes that can be mined to improve the detection and management of cancer. Beyond cell-free DNA (cfDNA), mutations have been detected in DNA associated with extracellular vesicles (EV-DNA). The genome-wide composition and structure of EV-DNA are poorly characterized, and it remains undecided whether circulating EVs are enriched in tumor signal compared to unfractionated cfDNA. Here, using whole genome sequencing from selected lung cancer patients with a high cfDNA tumor content (>5%), we determined that the tumor fraction and heterogeneity are comparable between DNA associated with EVs and matched plasma cfDNA. DNA in EV fractions, obtained with standardized size-exclusion chromatography, are comprised of short [~]150-180 bp fragments and long >1000 bp fragments that are poor in tumor signal. Other fractions only exhibit short fragments with similar tumor DNA content. The composition in bases at the end of EV-DNA fragments, as well as their fragmentation patterns are similar to plasma cfDNA. Mitochondrial DNA is relatively enriched in EV fractions. Our results highlight that cfDNA in plasma is of dual nature, either bound to proteins (including the nucleosome) but also associated to EV. cfDNA associated to small EV (including exosomes) is however not preferentially enriched in tumor signal.

genomics↗

bioRxiv2021

The effect of pre-analytical and physiological variables on cell-free DNA fragmentation.

Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, pre-analytic and physiological differences between individuals on fragmentomic analysis are poorly defined. We analyzed the impact of collection tube, plasma processing time and physiology on the size distribution of cfDNA, their genome-wide representation and sequence diversity at the cfDNA fragment-ends using shallow Whole Genome Sequencing. We observed that using different stabilizing collection tubes, or processing times does not affect the cfDNA fragment sizes, but can impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication and chronic conditions) minimally influenced the outcome of fragmentomic methods. Our results highlight that fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.

genomics↗

bioRxiv2021

Unbiased and UMI-informed sequencing of cell-free miRNAs at single-nucleotide resolution

Terminal nucleotidyl transferases are enzymes that add non-templated nucleotides to RNA molecules. In the case of microRNAs, this process was shown to be functionally relevant for their maturation process and generation of isomiRs with non-canonical mRNA targets. Deconvolution of these posttranscriptional modifications is challenging in particular for extracellular miRNAs that are considered as a target for minimally-invasive diagnostics. Massively parallel RNA sequencing is the only method that can truthfully reveal isomiR diversity in biological samples and determine relative quantities. Improvements aside, current small RNA sequencing strategies remain imprecise. We developed IsoSeek that diverges from these methods by making use of randomized 5- and 3-adapters combined with a 10N unique molecular identifier (UMI). Using synthetic miRNA and isomiR spike-in sets and testing depletion and RNA competition strategies in 7 sequencing rounds of >100 samples, we rigorously optimized and validated the technical accuracy of the IsoSeek method. In genetically-altered HEK293, we characterized the terminal uridylase (TUT4/TUT7) dependent miRNA uridylome and discovered extensive uridylation of disease-associated miRNAs. Notably, 3-uridylated isomiR profiles of plasma extracellular vesicles (EVs) rely on UMI-correction. Thus, IsoSeek advances our knowledge of cell-free miRNAs and supports development into non-invasive biomarkers.

genomics↗