Biology subjects

Dyrskjoet, L.

Publications and source records attributed to Dyrskjoet, L..

3 recordsLinked to original sources

bioRxiv2023

Error-corrected deep targeted sequencing of circulating cell-free DNA from colorectal cancer patients for sensitive detection of circulating tumor DNA

IntroductionCirculating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed UMIseq, a fixed-panel deep-targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. MethodsUMIseq features UMI-mediated error correction, exclusion of mutations related to clonal hematopoiesis, a panel of normals for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n=364) and healthy individuals (n=61). ResultsUMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AF) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while in the validation cohort, it was 70%. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. Detection of ctDNA was associated with recurrence (p =0.08). ConclusionUMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

cancer biology↗

bioRxiv2022

Improved Protocol for Single Nucleus RNA-sequencing of Frozen Human Bladder Tumor Biopsies

This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection; sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed a combination of IgePal lysis buffer, tissue dissection by sectioning and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well described clinical and histopathological information and known clinical outcomes with snRNA-seq.

cancer biology↗

bioRxiv2020

Transcriptome-wide profiles of circular RNA and RNA binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA binding proteins (RBPs), however, little is known about the prevalence and strength of these interactions in different biological contexts. Here, we comprehensively evaluate the interplay between circRNAs and RBPs in the ENCODE cell lines, HepG2 and K562, by profiling the expression of circRNAs in fractionated total RNA-sequencing samples and analyzing binding sites of 150 RBPs in large eCLIP data sets. We show that KHSRP binding sites are enriched in flanking introns of circRNAs in both HepG2 and K562 cells, and that KHSRP depletion affects circRNA biogenesis. Additionally, we show that exons forming circRNAs are generally enriched with RBP binding sites compared to non-circularizing exons. To detect individual circRNAs with regulatory potency, we computationally identify circRNAs that are highly covered by RBP binding sites and experimentally validate circRNA-RBP interactions by RNA immunoprecipitations. We characterize circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, which is covered with GRWD1 binding sites. We confirm that circCDYL binds GRWD1 in vivo and functionally characterizes the effect of circCDYL-GRWD1 interactions on target genes in HepG2. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g. TP53 and MYC. Finally, we show that elevated levels of highly RBP-covered circRNAs, including circCDYL, are associated with overall survival of bladder cancer patients. Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

genomics↗