Biology subjects

Nuytens, J.

Publications and source records attributed to Nuytens, J..

8 recordsLinked to original sources

bioRxiv2022

Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed by computational detection tools. During the last decade, a plethora of such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools were used and detected over 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were empirically validated using three orthogonal methods. Generally, tool-specific precision values are high and similar (median of 98.8%, 96.3%, and 95.5% for qPCR, RNase R, and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant tool differentiators. Furthermore, we demonstrate the complementarity of tools through the increase in detection sensitivity by considering the union of highly-precise tool combinations while keeping the number of false discoveries low. Finally, based on the benchmarking results, recommendations are put forward for circRNA detection and validation.

genomics↗

bioRxiv2021

mTOR inhibition enhances delivery and activity of antisense oligonucleotides in uveal melanoma cells

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Due to a lack of effective treatments, patients with metastatic disease have a median survival time of 6-12 months. We recently demonstrated that the SAMMSON long non-coding RNA (lncRNA) is essential for uveal melanoma cell survival and that antisense oligonucleotide (ASO)-mediated silencing of SAMMSON impaired cell viability and tumor growth in vitro and in vivo. By screening a library of 2911 clinical stage compounds, we identified the mTOR inhibitor GDC-0349 to synergize with SAMMSON inhibition in UM. Mechanistic studies revealed that mTOR inhibition enhanced uptake and reduced lysosomal accumulation of lipid complexed SAMMSON ASOs, improving SAMMSON knockdown and further decreasing UM cell viability. We found mTOR inhibition to also enhance target knockdown in other cancer cell lines as well as normal cells when combined with lipid nanoparticle complexed or encapsulated ASOs or small interfering RNAs (siRNAs). Our results are relevant to nucleic acid treatment in general and highlight the potential of mTOR inhibition to enhance ASO and siRNA mediated target knockdown.

cancer biology↗

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↗

bioRxiv2021

Custom long non-coding RNA capture enhances detection sensitivity in different human sample types

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

genomics↗

bioRxiv2019

Charting extracellular transcriptomes in The Human Biofluid RNA Atlas

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on plasma or serum, other biofluids may contain more informative RNA molecules, depending on the type of disease. Here, we present an unprecedented atlas of messenger, circular and small RNA transcriptomes of a comprehensive collection of 20 different human biofluids. By means of synthetic spike-in controls, we compared RNA content across biofluids, revealing a more than 10 000-fold difference in RNA concentration. The circular RNA fraction is increased in nearly all biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. In addition, a subset of biofluids, including stool, sweat, saliva and sputum, contains high levels of bacterial RNAs. Our atlas enables a more informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Taken together, our results reveal novel insights in the RNA content of human biofluids and may serve as a valuable resource for future biomarker studies. All spike-normalized data is publicly available in the R2 web portal and serve as a basis to further explore the RNA content in biofluids.

genomics↗

bioRxiv2019

The RNA Atlas, a single nucleotide resolution map of the human transcriptome

The human transcriptome consists of various RNA biotypes including multiple types of non-coding RNAs (ncRNAs). Current ncRNA compendia remain incomplete partially because they are almost exclusively derived from the interrogation of small- and polyadenylated RNAs. Here, we present a more comprehensive atlas of the human transcriptome that is derived from matching polyA-, total-, and small-RNA profiles of a heterogeneous collection of nearly 300 human tissues and cell lines. We report on thousands of novel RNA species across all major RNA biotypes, including a hitherto poorly-cataloged class of non-polyadenylated single-exon long non-coding RNAs. In addition, we exploit intron abundance estimates from total RNA-sequencing to test and verify functional regulation by novel non-coding RNAs. Our study represents a substantial expansion of the current catalogue of human ncRNAs and their regulatory interactions. All data, analyses, and results are available in the R2 web portal and serve as a basis to further explore RNA biology and function.

genetics↗

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↗

bioRxiv2017

Targeted genomic screen reveals focal long non-coding RNA copy number alterations in cancer

The landscape of somatic copy-number alterations (SCNAs) affecting long non-coding RNAs (lncRNAs) in human cancer remains largely unexplored. While the majority of lncRNAs remains to be functionally characterized, several have been implicated in cancer development and metastasis. Considering the plethora of lncRNAs genes that is currently reported, it is conceivable that several lncRNAs might function as oncogenes or tumor suppressor genes.\n\nWe devised a strategy to detect focal lncRNA SCNAs using a custom DNA microarray platform probing 20 418 lncRNA genes. By screening a panel of 80 cancer cell lines, we detected numerous focal aberrations targeting one or multiple lncRNAs without affecting neighboring protein-coding genes. These focal aberrations are highly suggestive for a tumor suppressive or oncogenic role of the targeted lncRNA gene. Although functional validation remains an essential step in the further characterization of the involved candidate cancer lncRNAs, our results provide a direct way of prioritizing candidate lncRNAs involved in cancer pathogenesis.

genetics↗