Patient-derived xenograft (PDX) and circulating tumor cell-derived explant (CDX) models are powerful methods for the study of human disease. In cancer research, these methods have been applied to ...multiple questions, including the study of metastatic progression, genetic evolution, and therapeutic drug responses. As PDX and CDX models can recapitulate the highly heterogeneous characteristics of a patient tumor, as well as their response to chemotherapy, there is considerable interest in combining them with next-generation sequencing to monitor the genomic, transcriptional, and epigenetic changes that accompany oncogenesis. When used for this purpose, their reliability is highly dependent on being able to accurately distinguish between sequencing reads that originate from the host, and those that arise from the xenograft itself. Here, we demonstrate that failure to correctly identify contaminating host reads when analyzing DNA- and RNA-sequencing (DNA-Seq and RNA-Seq) data from PDX and CDX models is a major confounding factor that can lead to incorrect mutation calls and a failure to identify canonical mutation signatures associated with tumorigenicity. In addition, a highly sensitive algorithm and open source software tool for identifying and removing contaminating host sequences is described. Importantly, when applied to PDX and CDX models of melanoma, these data demonstrate its utility as a sensitive and selective tool for the correction of PDX- and CDX-derived whole-exome and RNA-Seq data.
This study describes a sensitive method to identify contaminating host reads in xenograft and explant DNA- and RNA-Seq data and is applicable to other forms of deep sequencing.
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Tumour hypoxia is associated with poor patient outcome and resistance to therapy. It is accompanied by widespread changes in gene expression mediated largely through the transcription factors ...HIF1/2/3α. Hypoxia impacts on multiple pathways throughout the cell and has widespread effects on phenotype. Here we use sample-specific annotation approaches to determine the changes in transcript architecture that arise as result of alternative splicing in hypoxic cells. Using
data generated from a time course in reduced oxygenation we identified genome-wide switching between coding and noncoding isoforms, including a significant number of components of the DNA damage response pathway. Notably, HDAC6, a master regulator of the cytotoxic response, and TP53BP1, which sits at the nexus of the double-strand break repair pathway, both underwent a marked transition towards an intron-retention pattern with a concomitant decline in protein levels. These transitions from coding to noncoding isoforms were recapitulated in a large and independent cohort of 499 colorectal samples taken from The Cancer Genome Atlas (TCGA). The set of altered genes was enriched for multiple components of the Fanconi Anaemia, nucleotide excision and double-strand break repair pathways, and together correlating with tumour status at last contact. Altogether, these data demonstrate a new role for hypoxia-driven alternative splicing in regulating DNA damage response, and highlight the importance of considering alternative splicing as a critical factor in our understanding of human disease.
In most patients with small-cell lung cancer (SCLC)-a metastatic, aggressive disease-the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of ...patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan-Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.
With the rapidly growing demand for DNA analysis, the need for storing and processing large-scale genome data has presented significant challenges. This paper describes how the Genome Analysis ...Toolkit (GATK) can be deployed to an elastic cloud, and defines policy to drive elastic scaling of the application. We extensively analyse the GATK to expose opportunities for resource elasticity, demonstrate that it can be practically deployed at scale in a cloud environment, and demonstrate that applying elastic scaling improves the performance to cost tradeoff achieved in a simulated environment.
Abstract
Background
Hypoxia occurs within the majority of solid tumors. It has multiple impacts on tumor biology including angiogenesis, suppression of immune reactivity, enhanced receptor tyrosine ...kinase signaling, down regulation of DNA repair pathways, promotion of pro-survival phenotypes and increased proclivity for invasion and metastasis. Levels of hypoxia vary between and within tumors, correlate with patient outcomes, and can lead to differences in response to therapy. Despite considerable advances, our understanding of the regulatory processes that control these diverse and heterogeneous changes in cancer phenotype remain incomplete. A better understanding of how they are mediated will inform strategies for personalized medicine and promise to substantially advance our understanding of tumor heterogeneity.
Methods
Until recently, techniques for RNA-expression profiling have lacked the resolution necessary to detect genome wide changes in splicing. We exploited the increased precision offered by deep sequencing to investigate genome-wide remodelling of transcript architectures in both cell line and tumor samples. Using novel computational strategies we generated sample-specific gene models from RNA sequencing data generated over a timecourse of HCT116 colorectal carcinoma cells following a switch to 1% oxygenation and used these data to identify widespread changes in splicing. We applied similar strategies to raw RNA-sequencing data derived from TCGA.
Results
We detected the expression of thousands of novel isoforms and a systematic, pathway-dependent switch to the expression of noncoding transcripts at multiple genes, including many constituents of the Fanconi Anemia, nucleotide excision- and double strand break repair pathways. Of particular note were HDAC6 and 53BP1: critical regulators of DNA damage response. Both loci exhibited a hypoxia-dependent expression of retained-intron transcripts and a concomitant decline in protein levels, thus providing a novel and unanticipated mechanism by which hypoxia can drive genetic instability. Predictions made using our cell line model were recapitulated in a large cohort of 458 colorectal carcinomas derived from TCGA. Validation of the cell line data revealed, for the first time, a strong signature of isoform switching in which a widespread transition to noncoding expression is associated with poor patient outcome. These data were derived from independent patient biopsies sequenced by others, thus demonstrating the robustness of our findings.
Conclusions
These data therefore have clear, substantial, and immediate implications both for our understanding of the basic aetiology of solid tumors and for the development of RNA-based signatures and biomarkers in diagnostics and personalized medicine.
Citation Format: Crispin Miller, Danish Memon, Keren Dawson, Christopher Smowton, Wei Xing, Caroline Dive. Programmatic re-splicing of the hypoxia transcriptome regulates the DNA damage response. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 80.
In this paper a new approach to API remoting for GPU virtualisation is described which aims to reduce the amount of trusted code involved in 3D rendering for guest VMs. To achieve this it uses a ...modular driver framework to export large proportions of complex 3D graphics drivers into the guest's domain. It further provides a secure graphical user interface to untrusted domains. The implementation of Xen3D is described, which remotes the Gallium graphics driver model, a system designed for the creation of highly modular graphics drivers, and serves as a proof of concept.
Zero-effort payments Smowton, Christopher; Lorch, Jacob R.; Molnar, David ...
Proceedings of the 2014 ACM International Joint Conference on Pervasive and Ubiquitous Computing,
09/2014
Conference Proceeding
This paper presents Zero-Effort Payments (ZEP), a seamless mobile computing system designed to accept payments with no effort on the customer's part beyond a one-time opt-in. With ZEP, customers need ...not present cards nor operate smartphones to convey their identities. ZEP uses three complementary identification technologies: face recognition, proximate device detection, and human assistance. We demonstrate that the combination of these technologies enables ZEP to scale to the level needed by our deployments.
We designed and built ZEP, and demonstrated its usefulness across two real-world deployments lasting five months of continuous deployment, and serving 274 customers. The different nature of our deployments stressed different aspects of our system. These challenges led to several system design changes to improve scalability and fault-tolerance.
Analysing cancer genomics in the elastic cloud Smowton, Christopher; Miller, Crispin; Xing, Wei ...
2015 15th IEEE/ACM International Symposium on Cluster, Cloud and Grid Computing,
05/2015
Conference Proceeding
With the rapidly growing demand for DNA analysis, the need for storing and processing large-scale genome data has presented significant challenges. This paper describes how the Genome Analysis ...Toolkit (GATK) can be deployed to an elastic cloud, and defines policy to drive elastic scaling of the application. We extensively analyse the GATK to expose opportunities for resource elasticity, demonstrate that it can be practically deployed at scale in a cloud environment, and demonstrate that applying elastic scaling improves the performance to cost tradeoff achieved in a simulated environment.
In this paper, we explore the benefits of automatically determining the degree of parallelism used to perform genetic mutation calling in a hybrid cloud environment. We propose algorithms to ...automatically control both the hiring of hybrid cloud resources and the selection of the degree of parallelism employed in analysis tasks executed against that cloud. Using the Broad Institute's Genome Analysis Toolkit as a case study, we then conduct profile-driven simulation studies to characterise the circumstances in which our algorithms are beneficial or deleterious compared to simple, conventional baseline algorithms. We find that there are a wide range of cloud workload scenarios where our algorithms outperform the baselines, and thereby argue that automatic control of cloud scaling and task parallelism, using techniques like those proposed, are likely to be beneficially applicable to real-world biocomputing.
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