29 research outputs found
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ViFi: accurate detection of viral integration and mRNA fusion reveals indiscriminate and unregulated transcription in proximal genomic regions in cervical cancer.
The integration of viral sequences into the host genome is an important driver of tumorigenesis in many viral mediated cancers, notably cervical cancer and hepatocellular carcinoma. We present ViFi, a computational method that combines phylogenetic methods with reference-based read mapping to detect viral integrations. In contrast with read-based reference mapping approaches, ViFi is faster, and shows high precision and sensitivity on both simulated and biological data, even when the integrated virus is a novel strain or highly mutated. We applied ViFi to matched genomic and mRNA data from 68 cervical cancer samples from TCGA and found high concordance between the two. Surprisingly, viral integration resulted in a dramatic transcriptional upregulation in all proximal elements, including LINEs and LTRs that are not normally transcribed. This upregulation is highly correlated with the presence of a viral gene fused with a downstream human element. Moreover, genomic rearrangements suggest the formation of apparent circular extrachromosomal (ecDNA) human-viral structures. Our results suggest the presence of apparent small circular fusion viral/human ecDNA, which correlates with indiscriminate and unregulated expression of proximal genomic elements, potentially contributing to the pathogenesis of HPV-associated cervical cancers. ViFi is available at https://github.com/namphuon/ViFi
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CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich's Ataxia.
Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in intron 1 of the frataxin (FXN) gene, leading to significant decreased expression of frataxin, a mitochondrial iron-binding protein. We previously reported that syngeneic hematopoietic stem and progenitor cell (HSPC) transplantation prevented neurodegeneration in the FRDA mouse model YG8R. We showed that the mechanism of rescue was mediated by the transfer of the functional frataxin from HSPC-derived microglia/macrophage cells to neurons/myocytes. In this study, we report the first step toward an autologous HSPC transplantation using the CRISPR-Cas9 system for FRDA. We first identified a pair of CRISPR RNAs (crRNAs) that efficiently removes the GAA expansions in human FRDA lymphoblasts, restoring the non-pathologic level of frataxin expression and normalizing mitochondrial activity. We also optimized the gene-editing approach in HSPCs isolated from healthy and FRDA patients' peripheral blood and demonstrated normal hematopoiesis of gene-edited cells in vitro and in vivo. The procedure did not induce cellular toxic effect or major off-target events, but a p53-mediated cell proliferation delay was observed in the gene-edited cells. This study provides the foundation for the clinical translation of autologous transplantation of gene-corrected HSPCs for FRDA
Mapping clustered mutations in cancer reveals APOBEC3 mutagenesis of ecDNA
Clustered somatic mutations are common in cancer genomes and previous analyses reveal several types of clustered single-base substitutions, which include doublet- and multi-base substitutions1–5, diffuse hypermutation termed omikli6, and longer strand-coordinated events termed kataegis3,7–9. Here we provide a comprehensive characterization of clustered substitutions and clustered small insertions and deletions (indels) across 2,583 whole-genome-sequenced cancers from 30 types of cancer10. Clustered mutations were highly enriched in driver genes and associated with differential gene expression and changes in overall survival. Several distinct mutational processes gave rise to clustered indels, including signatures that were enriched in tobacco smokers and homologous-recombination-deficient cancers. Doublet-base substitutions were caused by at least 12 mutational processes, whereas most multi-base substitutions were generated by either tobacco smoking or exposure to ultraviolet light. Omikli events, which have previously been attributed to APOBEC3 activity6, accounted for a large proportion of clustered substitutions; however, only 16.2% of omikli matched APOBEC3 patterns. Kataegis was generated by multiple mutational processes, and 76.1% of all kataegic events exhibited mutational patterns that are associated with the activation-induced deaminase (AID) and APOBEC3 family of deaminases. Co-occurrence of APOBEC3 kataegis and extrachromosomal DNA (ecDNA), termed kyklonas (Greek for cyclone), was found in 31% of samples with ecDNA. Multiple distinct kyklonic events were observed on most mutated ecDNA. ecDNA containing known cancer genes exhibited both positive selection and kyklonic hypermutation. Our results reveal the diversity of clustered mutational processes in human cancer and the role of APOBEC3 in recurrently mutating and fuelling the evolution of ecDNA
Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies
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Alignment Methods for Optical Maps
Optical mapping is a DNA physical mapping technique that can measure large-scale structural variation in genomes and enables more accurate completion of genome assemblies. Particularly, we focus on the case where optical mapping is used to complete genome assembly on pre-identified segments such as may be found in a breakpoint graph. In this work we study methods for aligning optical map contigs with reference genome segments. This work introduces a novel method for optical map alignment that outperforms existing methods for aligning breakpoint graph reference segments to assembled optical map contigs. Also discussed are some additional modifications that can be made to the method
Alignment Methods for Optical Maps
Optical mapping is a DNA physical mapping technique that can measure large-scale structural variation in genomes and enables more accurate completion of genome assemblies. Particularly, we focus on the case where optical mapping is used to complete genome assembly on pre-identified segments such as may be found in a breakpoint graph. In this work we study methods for aligning optical map contigs with reference genome segments. This work introduces a novel method for optical map alignment that outperforms existing methods for aligning breakpoint graph reference segments to assembled optical map contigs. Also discussed are some additional modifications that can be made to the method
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Reconstructing and Profiling Extrachromosomal DNA
Circular extrachromosomal DNA (ecDNA) are a genomic lesion occurring in tumors, and represent a foundational, growing frontier in cancer biology. The discovery that focal amplifications exist in multiple topologies and arise by different mechanisms has enabled cancer researchers to study the consequences of different types of focal amplification – revealing that focal amplifications like ecDNA lead to worse patient survival. Consequently, there is an urgent need for bioinformatic methods and tools to study these focal amplifications, particularly ecDNA. This thesis describes novel tools and methods which can be used to study ecDNA, and other focal amplifications. It also demonstrates how those tools and methods can be used to profile focal amplifications across different cancer types, ultimately revealing novel biology about the structure, function and genesis of ecDNA in different contexts. I first present two methods, AmpliconReconstructor (AR) and FaNDOM, which incorporate optical mapping data to resolve the structures of ecDNA and other focal amplifications. AR incorporates both optical mapping and NGS data and builds upon a prior method for ecDNA detection with NGS data, AmpliconArchitect (AA). FaNDOM utilizes optical mapping solely and enables the rapid characterization of large structural variants using assembled OM contigs or individual OM molecules. I also describe the landscape of ecDNA in oropharyngeal squamous cell carcinoma, demonstrating that both human and hybrid human-viral ecDNA exist and are associated with distinct patterns of transcriptional splicing. Visualizations of the rearranged ecDNA structures and overlaid transcription-level data reveal the overexpression of genes carried on ecDNA. Lastly, I describe the genesis of ecDNA in Barrett’s esophagus, the precursor tissue of esophageal adenocarcinoma. We utilized methods for profiling ecDNA, such as AmpliconClassifier, to demonstrate that ecDNA exist in pre-cancerous tissue, are associated with worse histology, that they are subsequently found again in cancer, and that they tend to undergo positive selection during the malignant transformation. These findings solidify ecDNA as a potent driver of cancer, and not an opportunistic passenger
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ViFi: accurate detection of viral integration and mRNA fusion reveals indiscriminate and unregulated transcription in proximal genomic regions in cervical cancer.
The integration of viral sequences into the host genome is an important driver of tumorigenesis in many viral mediated cancers, notably cervical cancer and hepatocellular carcinoma. We present ViFi, a computational method that combines phylogenetic methods with reference-based read mapping to detect viral integrations. In contrast with read-based reference mapping approaches, ViFi is faster, and shows high precision and sensitivity on both simulated and biological data, even when the integrated virus is a novel strain or highly mutated. We applied ViFi to matched genomic and mRNA data from 68 cervical cancer samples from TCGA and found high concordance between the two. Surprisingly, viral integration resulted in a dramatic transcriptional upregulation in all proximal elements, including LINEs and LTRs that are not normally transcribed. This upregulation is highly correlated with the presence of a viral gene fused with a downstream human element. Moreover, genomic rearrangements suggest the formation of apparent circular extrachromosomal (ecDNA) human-viral structures. Our results suggest the presence of apparent small circular fusion viral/human ecDNA, which correlates with indiscriminate and unregulated expression of proximal genomic elements, potentially contributing to the pathogenesis of HPV-associated cervical cancers. ViFi is available at https://github.com/namphuon/ViFi
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EcSeg: Semantic Segmentation of Metaphase Images Containing Extrachromosomal DNA.
Oncogene amplification is one of the most common drivers of genetic events in cancer, potently promoting tumor development, growth, and progression. The recent discovery that oncogene amplification commonly occurs on extrachromosomal DNA, driving intratumoral genetic heterogeneity and high copy number owing to its non-chromosomal mechanism of inheritance, raises important questions about how the subnuclear location of amplified oncogenes mediates tumor pathogenesis. Next-generation sequencing is powerful but does not provide spatial resolution for amplified oncogenes, and new approaches are needed for accurately quantifying oncogenes located on ecDNA. Here, we introduce ecSeg, an image analysis tool that integrates conventional microscopy with deep neural networks to accurately resolve ecDNA and oncogene amplification at the single cell level