Biology subjects

Boyle, S.

Publications and source records attributed to Boyle, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The effects of estrogen exposure on survival, growth, and fecundity of Daphnia magna

High concentrations of steroidal hormone compounds are a growing source of concern for environmental pollution in aquatic ecosystems. In this study, we examine the effects of two estrogenic compounds (estriol and 17-ethinylestradiol) on fitness traits in the aquatic microcrustacean, Daphnia magna, a key bioindicator species for toxicology studies. The impacts were compared of two forms representing a natural and synthetic estrogenic compound. Growth and reproduction traits were assayed by exposing Daphnia to each estrogen type at four concentrations reflecting potential environmental exposure conditions up to acute toxicity levels (ranging from 0.1 - 50 {micro}g/L). Assaying the effects at a variety of concentrations is important given that it is known that hormone exposures can often result in non-monotonic responses. Both forms of estrogen impact a subset of the traits assessed, in some cases leading to beneficial changes and others causing harm. Estriol, the naturally-occurring estrogen, and EE2, the synthetic version, at high doses shift fitness traits in opposite directions such as adult growth rate as do at low doses for fecundity. In conclusion, our results support the need to assay a wide array of traits using multiple forms of steroidal hormones at a range of doses in order to assess non-monotonic patterns and their impact on an organismal fitness. In particular, assays that extend beyond the conventional measurements of lethality during acute exposure windows will be essential for understanding the impact of increased levels of hormone pollution on aquatic organisms and ecosystem health.

pharmacology and toxicology↗

bioRxiv2026

Loop extrusion dynamics and cooperative activation independently regulate enhancer-driven transcription

How enhancers transmit their activation signals to target promoters is a fundamental unanswered question. Gene activation by distal enhancers is affected by the three-dimensional organisation of chromatin driven by cohesin-mediated loop extrusion, but how the dynamic loop extrusion cycle shapes transcriptional outcomes is not fully understood. In addition, genes are typically regulated by multiple enhancers and cases of cooperativity between enhancers have been observed that depend on the relative position of enhancers and gene. The reason for such context-dependent cooperativity is unknown, and whether it is linked to loop extrusion has not been explored. Here, we use synthetic gene activation to show that NIPBL depletion, which decreases loop extrusion rate, reduces long-range activation in a dose-dependent manner. In contrast, WAPL depletion, which reduces cohesin turnover, can both increase and decrease distal gene activation depending on the presence of adjacent CTCF binding sites. These effects on gene activation correlate with the impact on chromatin contacts. Depletion of the co-activator BRD4 also affects long-range activation, but not by changing chromatin contacts. Synthetic activation from two locations results in super-additive responses, which can be predicted by an independently determined response model. These predictions hold for dual activation from proximal and/or distal sites, and with perturbed NIPBL, WAPL, or BRD4. Distal activation therefore appears to be equivalent in nature to proximal activation, and context-dependent cooperativity can arise simply from different level of activation inputs operating on a non-linear response function.

molecular biology↗

bioRxiv2025

DICER1 syndrome mutations lead to a gain of 3p-miRNA function, HERVH activity and increased metastatic potential

The DICER1 gene is mutated in cancer, including DICER1 syndrome, a rare tumour predisposition syndrome. Cancer-associated hotspot mutations have been reported in both catalytic domains of DICER1 and are predicted to disrupt miRNA biogenesis. To understand how these hotspot mutations contribute to cancer development, we have generated cell lines harbouring single amino acid substitutions within the catalytic RNase IIIa (S1344L) or RNase IIIb (D1709N) domains of the endogenous DICER1 gene. We show that both mutations result in a widespread loss of 5p miRNAs, and, unexpectedly, an increase in 3p passenger strands loading into AGO2. The shared similarities between both mutants can be attributed to the structural proximity of the S1344 residue to the RNase IIIb catalytic centre. Functionally, we found that changes in the repertoire of miRNAs loaded into AGO2 result in altered gene expression, impacting critical pathways for cancer development, including metastatic potential. Additionally, our results indicate that inactivating the processing activity of DICER1 does not result in genomic instability. Instead, mutations cause specific upregulation of human endogenous retrovirus H (HERVH) through miRNA-independent mechanisms, suggesting that both canonical and non-canonical DICER1 functions are important to understand DICER1 syndrome.

molecular biology↗

bioRxiv2025

Chromosome Ageing Occurs at the Primordial Follicle Stage in both Mouse and Human Oocytes

Inherited aneuploidy is one of the most common causes of human genetic disease affecting approximately 20% of all human embryos and is the major cause of non-conception in older women. Inherited aneuploidies typically arise from meiotic chromosome segregation errors and are strongly affected by maternal age. Cohesin, a protein complex that mediates cohesion between sister chromatids, becomes depleted from oocyte chromosomes with age in mice, potentially contributing to the high rate of aneuploidy in older mouse oocytes. However, it remains unclear at which stage of oogenesis ageing affects chromatid cohesion in mice and whether ageing similarly affects cohesin in human oocytes. Here we use fluorescence in situ hybridisation to assess at which stages of oogenesis ageing alters chromatid separation in mice and in humans. We show that chromatid separation increases with age while oocytes remain dormant in primordial follicles in both mice and humans and that this effect of age on chromatid separation is detectable in fertile women of child-bearing age. Furthermore, we show that this age-dependent increase in chromatid separation is accompanied by age-dependent depletion of the acetylated SMC3-marked cohesive subpopulation of cohesin from oocytes at the dormant primordial follicle stage in both mice and humans. These data suggest that ageing impacts on oocyte chromosomes while they remain dormant in primordial follicles, and that this aspect of oocyte chromosome ageing is shared in both mice and humans. Improving our understanding of these pathways may allow strategies to slow or prevent this aspect of oocyte ageing to be developed.

developmental biology↗

bioRxiv2025

Embryonic stem cells do not have a globally disrupted higher-order chromatin fibre structure

Embryonic stem cells (ESCs) are thought to maintain pluripotency through global hyper-transcription, linked to an "open" chromatin structure. To investigate this idea, we analyzed higher-order chromatin fibres from mouse ESCs, their differentiated progenitors, and terminally differentiated mouse NIH3T3 fibroblasts. Bulk chromatin composition including protein:DNA ratio and nucleosome repeat length varied little between the cell types, but surprisingly biophysical analyses such as linker histone FRAP, hydrodynamic sedimentation and nuclease sensitivity also showed no significant differences in the conformation of purified higher-order chromatin fibres. To better evaluate the structure of higher-order chromatin fibres observed in cells, we developed a novel technique called SPOCC (Sedimentation Properties of Cross-Linked Chromatin). This approach revealed that ESCs and differentiated cells share similar bulk higher-order chromatin fibre structures, whilst ESCs have a slightly more disrupted structure than NIH3T3 cell chromatin. These results indicate that ESC transcriptional activity and plasticity are not driven by a fundamentally "open" higher-order chromatin conformation.

molecular biology↗

bioRxiv2024

TPR is required for cytoplasmic chromatin fragment formation during senescence

During oncogene-induced senescence there are striking changes in the structure of the nucleus and the organisation of heterochromatin. This is accompanied by activation of a pro-inflammatory gene expression programme - the senescence associated secretory phenotype (SASP) - driven by transcription factors such as NF-{kappa}B. Here we show that TPR, a protein of the nuclear pore complex basket, is required for the very early activation of NF-{kappa}B signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-{kappa}B nuclear import. We show that TPR is required for the activation of TBK1 signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. These cytoplasmic chromatin fragments appear to lack nuclear pore components. Our data suggest that TPR at the nuclear pore is involved in the loss of structural integrity of the nuclear periphery during senescence. We propose that this acts as a trigger for activation of cytoplasmic nucleic acid sensing, NF-{kappa}B signalling, and activation of the SASP, during senescence.

cell biology↗

bioRxiv2023

Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and physical proximity

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation by Capture-C, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent but are influenced by enhancer-bound protein complexes whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

genomics↗

bioRxiv2023

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops

Polycomb-repressive complex 1 (PRC1) has a strong influence on 3D genome organization, mediating local chromatin compaction as well as localized and chromosome-wide clustering of target loci. Several subunits of PRC1 have been shown to have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexandiol, which disrupts liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation combined with deep sequencing analyses, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci - at megabase and tens of megabase length scales - can be reversibly disrupted by the addition and subsequent removal of 1,6-hexandiol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to the reduction of PRC1 binding following 1,6-hexandiol treatment as the addition of 2,5-hexandiol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules, characteristic of liquid-liquid phase separation, have a role in polycomb-mediated genome organization.

molecular biology↗

bioRxiv2022

Ultra-long-range interactions between active regulatory elements

Contacts between enhancers and promoters are thought to relate to their ability to activate transcription. Investigating mechanisms that drive such chromatin interactions is therefore important for understanding gene regulation. Here, we have determined contact frequencies between millions of pairs of cis-regulatory elements from chromosome conformation capture datasets and analysed a collection of hundreds of DNA-binding factors for binding at regions of enriched contacts. This analysis revealed enriched contacts at sites bound by many factors associated with active transcription. We show that active regulatory elements, independent of cohesin and polycomb, interact with each other across distances of 10s of megabases in vertebrate and invertebrate genomes and that interactions correlate and change with activity. However, these ultra-long-range interactions are not dependent on RNA polymerase II transcription or several transcription cofactors. We propose that long-range interactions between cis-regulatory elements are driven by three distinct mechanisms - cohesin-mediated loop extrusion, polycomb contacts, and association between active regions.

genomics↗

bioRxiv2022

Multi-site Performance Evaluation of the cobas 5800 System and Comparison to the cobas 6800/8800 Systems for Quantitative Measurement of HBV, HCV, and HIV-1 Viral Load.

BackgroundThe cobas(R) 5800 System ("cobas 5800") is a new low to mid-throughput PCR-based nucleic acid testing system which performs both qualitative and quantitative testing, including viral load (VL) determination. cobas 5800 shares numerous design elements and technical characteristics with the existing cobas 6800/8800 Systems. Study DesignWe compared HBV, HCV, and HIV-1 VL results from cobas 5800 in three different laboratories to those from the same specimens tested on a cobas 6800 system. We also assessed cobas 5800 assay reproducibility by repetitive testing of specimens with VL close to values used as thresholds for patient management or classification. ResultsThe correlation between VL measurements generated using cobas 5800 vs 6800 was extremely high, with r2 correlation coefficients between 0.990 and 0.999 for the three targets at the different sites. The slope of the Deming regression line ranged from 0.994 (HBV, site 3) to 1.025 (HIV-1, site 1). The standard deviation values ranged from 0.04 to 0.19 log10 IU/mL for HBV, 0.05 to 0.31 log10 IU/mL for HCV, and 0.05 to 0.31 log10 copies/mL for HIV-1. In general, variability was higher at lower VL. Between 98.6% and 100% of results fell within the allowable total difference zone that defines expected variability on the existing 6800/8800 system. ConclusionsThis multi-site comparison study demonstrates equivalent performance of the new cobas 5800 system compared to cobas 6800. This establishes cobas 5800 as a new option for low to mid-throughout laboratories seeking to optimize efficiency of their viral molecular testing.

microbiology↗

bioRxiv2022

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional condensates, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harboring distinct ecDNA species (EGFR, MYC, PDGFR). We found no evidence that ecDNA cluster with one another or closely interact with transcriptional condensates. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy was similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with the cellular transcriptional machinery.

genetics↗

bioRxiv2021

Transcription modulates chromatin dynamics and locus configuration sampling

In living cells the 3D structure of gene loci is dynamic, but this is not revealed by 3C and FISH experiments in fixed samples, leaving a significant gap in our understanding. To overcome these limitations we applied the "highly predictive heteromorphic polymer" (HiP-HoP) model, validated by experiments, to determine chromatin fibre mobility at the Pax6 locus in three mouse cell lines with different transcription states. While transcriptional activity minimally affects the movement of 40 kbp regions, we observed that the motion of smaller 1 kbp regions depends strongly on local disruption to chromatin fibre structure marked by H3K27 acetylation. This also significantly influenced locus configuration dynamics by modulating promoter-enhancer loops associated with protein bridging. Importantly these simulations indicate that chromatin dynamics are sufficiently fast to sample all possible conformations of loci within minutes, generating wide dynamic variability of gene loci structure within single cells. Experiments inhibiting transcription change chromatin fibre structure subtly, yet we predict they should substantially affect mobility. This combination of simulation and experimental validation provide a novel insight and mechanistic model to explain how transcriptional activity influences chromatin structure and gene dynamics.

biophysics↗

bioRxiv2021

Nuclear Lamina binds the EBV genome during latency and regulates viral gene expression

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus. AUTHOR SUMMARYEpstein-Barr virus (EBV) is a common herpesvirus that establishes a lifelong latent infection in a small fraction of B cells of the infected individuals. In most cases, EBV infection is asymptomatic; however, especially in the context of immune suppression, EBV latent infection is associated with several malignancies. In EBV+ cancer cells, latent viral gene expression plays an essential role in sustaining the cancer phenotype. We and others have established that epigenetic modifications of the viral genome are critical to regulating EBV gene expression during latency. Understanding how the EBV genome is epigenetically regulated during latent infection may help identify new specific therapeutic targets for treating EBV+ malignancies. The nuclear lamina is involved in regulating the composition and structure of the cellular chromatin. In the present study, we determined that the nuclear lamina binds the EBV genome during latency, influencing viral gene expression. Depleting one component of the nuclear lamina, lamin A/C, increased the expression of latent EBV genes associated with cellular proliferation, indicating that the binding of the nuclear lamina with the viral genome is essential to control viral gene expression in infected cells. Our data show for the first time that the nuclear lamina may be involved in the cellular response against EBV infection by restricting viral gene expression.

microbiology↗

bioRxiv2021

Neurotensin neurons in the central extended amygdala control energy balance

Overeating and a sedentary life style are major causes of obesity and related metabolic disorders. Identification of the neurobiological processes that regulate energy balance will facilitate development of interventions for these disorders. Here we show that the Neurotensin-expressing neurons in the mouse IPAC (IPACNts), a nucleus of the central extended amygdala, bidirectionally coordinate hedonic feeding and physical activity, thereby regulating energy balance, metabolic processes and bodyweight. IPACNts are preferentially activated by consumption of highly palatable food or exposure to its taste and smell. Activating IPACNts promotes food intake in a palatability-dependent manner and decreases locomotion. Conversely, inhibiting IPACNts selectively reduces palatable food intake and dramatically enhances physical activity and energy expenditure, and in parallel stimulates physiological responses that oppose diet-induced obesity and metabolic dysfunctions. Thus, a single neuronal population, Neurotensin-expressing neurons in the IPAC, acts to control obesogenic and leptogenic processes by synergistically coordinating energy intake and expenditure with metabolism.

neuroscience↗

bioRxiv2020

The connector between the kinase and RNase domains of IRE1α transmits the conformational change that underlies ER stress-induced activation.

The unfolded protein response (UPR) plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1 is a major transducer that conveys endoplasmic reticulum (ER) stress to biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that upon sensing unfolded proteins via its ER luminal domain, IRE1 dimerizes and oligomerizes (often visualized as clustering), and then trans-autophosphorylates. The IRE1 kinase activity is required for activation of its RNase effector domain and for clustering of IRE1. It is not yet clear if IRE1 clustering is a platform for the RNase activity, or if the two represent distinct biological functions. Here, we uncover a previously unrecognized role for helix K between IRE1 kinase and RNase domains in conveying critical conformational changes. Using mutants within this inter-domain helix, we show for the first time that: 1) distinct substitutions (specifically, of Leu827) selectively affect oligomerization, RNase activity, and, unexpectedly, the kinase activity of IRE1; 2) RNase activation can be uncoupled from IRE1 oligomerization, and phosphorylation of S729 marks the former but not the latter; 3) The nature of residue 827 determines the conformation that the IRE1 protein adopts, leading to different patterns of biochemical activities. In summary, this work reveals a previously unappreciated role for the inter-domain helix as a pivotal conduit for attaining the stress-responsive conformation of IRE1.

biochemistry↗

bioRxiv2019

A Central Role for Canonical PRC1 in Shaping the 3D Nuclear Landscape

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus to form observable bodies; a conformation which is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organisation and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb Repressive Complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are seperable events. These findings provide important insights into the function of PRC1, whilst highlighting the complexity of this regulatory system. HighlightsO_LILoss of RING1B substantially disrupts nuclear architecture. C_LIO_LIPRC1 mediated looping can occur at a Mb scale and is independent of CTCF. C_LIO_LIPolycomb mediated looping is driven by canonical PRC1 complexes. C_LIO_LIMultimeric PRC1-mediated interactions occur in vitro and in vivo. C_LIO_LIDisruption of PRC1-mediated looping is independent of gene activation. C_LI

molecular biology↗

bioRxiv2019

EBV latent membrane protein 1 induces lipogenesis and confers sensitivity to inhibition of fatty acid synthase in B cells.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkins and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitts lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1 and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B-cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1 and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could potentially be used in the treatment of LMP1+ EBV associated malignancies by targeting a LMP1-specific dependency on lipogenesis. ImportanceDespite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of viral-associated malignancies. Non-cancerous cells preferentially obtain fatty acids from dietary sources whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1 and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis could be an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

microbiology↗

bioRxiv2019

Detection of ctDNA from dried blood spots after DNA size selection

Recent advances in the research and clinical applications of circulating tumour DNA (ctDNA) is limited by practical considerations of sample collection. Whole genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations, fragment size patterns, and other genomic features. We hypothesised that low-depth WGS data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may be effective to remove contaminating genomic DNA (gDNA) from small volumes of blood. There are practical advantages to using dried blood spots as these are easier to collect, facilitate serial sampling, and support novel study designs in prospective human studies, animal models and expand the utilisation of archival samples by the removal of gDNA in small volumes. We therefore developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots. Analysing a dried blood spot of 50L frozen whole blood from a patient with melanoma, we identified ctDNA based on tumour-specific somatic copy-number alterations, and found a fragment size profile similar to that observed in plasma DNA processed by traditional methods. We extended this approach to detect tumour-derived cell-free DNA in a dried blood spot from a mouse xenograft model and were able to identify ctDNA from the originally grafted ascites. Together, our data suggests that ctDNA can be detected and monitored in dried blood spots. This will enable new approaches for sample collection from patients and in vivo models.

cancer biology↗