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Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germline functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming.
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Metilação de DNA , Embrião não Mamífero/metabolismo , Peixe-Zebra/genética , Animais , Epigênese Genética , Feminino , Fertilização , Masculino , Oócitos/metabolismo , Espermatozoides/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKRWT) or PKR with a point mutation in each dsRNA-binding motif (PKRRM). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKRWT samples after PA treatment, but not in the PKRRM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5'-triphosphate enhanced PKR activity compared with the activity with a 5'-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.
Assuntos
RNA Nucleolar Pequeno/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo , Animais , Células CHO , Extratos Celulares , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Imunoprecipitação , Camundongos , Ácido Palmítico/farmacologia , Reprodutibilidade dos Testes , Estresse Fisiológico/efeitos dos fármacosRESUMO
Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with â¼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3' UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.
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Caenorhabditis elegans/genética , Perfilação da Expressão Gênica , Genoma Helmíntico , RNA de Cadeia Dupla/genética , Transcriptoma , Regiões 3' não Traduzidas , Adenosina Desaminase/metabolismo , Animais , Sequência de Bases , Perfilação da Expressão Gênica/métodos , Íntrons , Dados de Sequência Molecular , Edição de RNARESUMO
Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at http://hci-exlovd.hci.utah.edu, with 77 re-evaluable variants.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Biologia Computacional/métodos , Substituição de Aminoácidos , Simulação por Computador , Bases de Dados Genéticas , Feminino , Predisposição Genética para Doença , Humanos , Mutação de Sentido Incorreto , Splicing de RNARESUMO
Because nucleosomes are widely replaced by protamine in mature human sperm, the epigenetic contributions of sperm chromatin to embryo development have been considered highly limited. Here we show that the retained nucleosomes are significantly enriched at loci of developmental importance, including imprinted gene clusters, microRNA clusters, HOX gene clusters, and the promoters of stand-alone developmental transcription and signalling factors. Notably, histone modifications localize to particular developmental loci. Dimethylated lysine 4 on histone H3 (H3K4me2) is enriched at certain developmental promoters, whereas large blocks of H3K4me3 localize to a subset of developmental promoters, regions in HOX clusters, certain noncoding RNAs, and generally to paternally expressed imprinted loci, but not paternally repressed loci. Notably, trimethylated H3K27 (H3K27me3) is significantly enriched at developmental promoters that are repressed in early embryos, including many bivalent (H3K4me3/H3K27me3) promoters in embryonic stem cells. Furthermore, developmental promoters are generally DNA hypomethylated in sperm, but acquire methylation during differentiation. Taken together, epigenetic marking in sperm is extensive, and correlated with developmental regulators.
Assuntos
Cromatina/metabolismo , Genes/genética , Nucleossomos/metabolismo , Espermatozoides/metabolismo , Metilação de DNA , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox/genética , Impressão Genômica , Genômica , Humanos , Masculino , MicroRNAs/genética , Família Multigênica/genética , Regiões Promotoras Genéticas , Protaminas/metabolismoRESUMO
Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. Here, we determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the D. melanogaster genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Metilação de DNA , Complexo Repressor Polycomb 1 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Obesity is a modifiable predisposition factor for postmenopausal breast cancer. This suggests a localized, reciprocal interaction between breast cancer cells and the surrounding mammary white adipose tissue. To investigate how breast cancer cells alter the composition and function of adipose tissue, we screened the secretomes of 10 human breast cancer cell lines for the ability to modulate the differentiation of adipocyte stem and progenitor cells. The screen identified an adipogenic modulator, zinc-alpha-2-glycoprotein (ZAG/AZGP1) that is secreted by triple-negative breast cancer (TNBC) cells. TNBC-secreted ZAG inhibits adipogenesis and instead induces the expression of fibrotic genes. Accordingly, depletion of ZAG in TNBC cells attenuates fibrosis in white adipose tissue and inhibits tumor growth. Further, high expression of ZAG is linked to poor prognosis in patients with TNBC but not in patients with other clinical subtypes of breast cancer. Our findings suggest a role of TNBC-secreted ZAG in promoting the transdifferentiation of adipocyte stem and progenitor cells into cancer-associated fibroblasts to support tumorigenesis. SIGNIFICANCE: Functional screening of breast cancer secretomes revealed that triple-negative breast cancer promotes fibrosis in the adipose tissue microenvironment by secreting zinc-alpha-2-glycoprotein and promoting the transdifferentiation of adipocyte stem cells into myofibroblasts.
Assuntos
Fibrose , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Feminino , Camundongos , Fibrose/metabolismo , Fibrose/patologia , Animais , Linhagem Celular Tumoral , Adipogenia , Adipócitos/metabolismo , Adipócitos/patologia , Glicoproteína Zn-alfa-2 , Microambiente Tumoral , Proteínas de Plasma Seminal/metabolismo , Proteínas de Plasma Seminal/genética , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologiaRESUMO
Obesity is a predisposition factor for breast cancer, suggesting a localized, reciprocal interaction between breast cancer cells and the surrounding mammary white adipose tissue. To investigate how breast cancer cells alter the composition and function of adipose tissue, we screened the secretomes of ten human breast cancer cell lines for the ability to modulate the differentiation of adipocyte stem and progenitor cells (ASPC). The screen identified a key adipogenic modulator, Zinc Alpha-2-Glycoprotein (ZAG/AZGP1), secreted by triple-negative breast cancer (TNBC) cells. TNBC-secreted ZAG inhibits adipogenesis and instead induces the expression of fibrotic genes. Accordingly, depletion of ZAG in TNBC cells attenuates fibrosis in white adipose tissue and inhibits tumor growth. Further, high expression of ZAG in TNBC patients, but not other clinical subtypes of breast cancer, is linked to poor prognosis. Our findings suggest a role of TNBC-secreted ZAG in promoting the transdifferentiation of ASPCs into cancer-associated fibroblasts to support tumorigenesis.
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PURPOSE: This study investigates a real-world multicenter cohort of patients with urinary tract cancer (UTC), with primary disease sites including the bladder, urethra, and upper tract, who enrolled for research molecular testing of their germline and tumor. The purpose of this study was to evaluate factors that could affect the likelihood of identifying a clinically actionable germline pathogenic variant (PV). METHODS: Patients with UTC were identified from 10 cancer institutes of the Oncology Research Information Exchange Network consortium. The data set comprised abstracted clinical data with germline and tumor genomic data, and comparative analyses were conducted. RESULTS: Clinically actionable germline PVs in cancer predisposition genes were identified in 16 (4.5%) of 354 patients. A higher proportion of patients with the urethra and the upper tract as the primary sites of disease had PVs with a prevalence of 11% (5/45), compared with only 3.6% (11/308) in those with the bladder as the primary site of disease (P = .04). There were no significant differences in markers of genomic instability (such as tumor mutational burden, microsatellite instability [MSI], and loss of heterozygosity, copy number, and chromosomal instability) between those with PVs and those without (P > .05). Of the PVs identified, 10 (62%) were in homologous recombination repair (HRR) genes, three (19%) in mismatch repair (MMR) genes, and three (19%) in genes associated with other pathways. CONCLUSION: Tissue-based assessment of genomic instability, such as MSI, does not reliably indicate germline PV. A comprehensive clinical germline testing approach that includes HRR genes in addition to MMR genes is likely to yield PVs in approximately one of 10 patients with nonbladder primary disease sites such as the upper tract and the urethra.
Assuntos
Sequenciamento do Exoma , Mutação em Linhagem Germinativa , Neoplasias Urológicas , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Neoplasias Urológicas/genética , Genômica , Predisposição Genética para Doença , Adulto , Idoso de 80 Anos ou mais , Estudos de CoortesRESUMO
Inbred mice are a useful tool for studying the in vivo functions of platelets. Nonetheless, the mRNA signature of mouse platelets is not known. Here, we use paired-end next-generation RNA sequencing (RNA-seq) to characterize the polyadenylated transcriptomes of human and mouse platelets. We report that RNA-seq provides unprecedented resolution of mRNAs that are expressed across the entire human and mouse genomes. Transcript expression and abundance are often conserved between the 2 species. Several mRNAs, however, are differentially expressed in human and mouse platelets. Moreover, previously described functional disparities between mouse and human platelets are reflected in differences at the transcript level, including protease activated receptor-1, protease activated receptor-3, platelet activating factor receptor, and factor V. This suggests that RNA-seq is a useful tool for predicting differences in platelet function between mice and humans. Our next-generation sequencing analysis provides new insights into the human and murine platelet transcriptomes. The sequencing dataset will be useful in the design of mouse models of hemostasis and a catalyst for discovery of new functions of platelets. Access to the dataset is found in the "Introduction."
Assuntos
Plaquetas/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genéticaRESUMO
Objectives: Using agile software development practices, develop and evaluate an architecture and implementation for reliable and user-friendly self-service management of bioinformatic data stored in the cloud. Materials and methods: Comprehensive Oncology Research Environment (CORE) Browser is a new open-source web application for cancer researchers to manage sequencing data organized in a flexible format in Amazon Simple Storage Service (S3) buckets. It has a microservices- and hypermedia-based architecture, which we integrated with Test-Driven Development (TDD), the iterative writing of computable specifications for how software should work prior to development. Relying on repeating patterns found in hypermedia-based architectures, we hypothesized that hypermedia would permit developing test "templates" that can be parameterized and executed for each microservice, maximizing code coverage while minimizing effort. Results: After one-and-a-half years of development, the CORE Browser backend had 121 test templates and 875 custom tests that were parameterized and executed 3031 times, providing 78% code coverage. Discussion: Architecting to permit test reuse through a hypermedia approach was a key success factor for our testing efforts. CORE Browser's application of hypermedia and TDD illustrates one way to integrate software engineering methods into data-intensive networked applications. Separating bioinformatic data management from analysis distinguishes this platform from others in bioinformatics and may provide stable data management while permitting analysis methods to advance more rapidly. Conclusion: Software engineering practices are underutilized in informatics. Similar informatics projects will more likely succeed through application of good architecture and automated testing. Our approach is broadly applicable to data management tools involving cloud data storage.
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Years after the discovery that Dicer is a key enzyme in gene silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in Caenorhabditis elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not necessary for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wild-type and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26-nucleotide small RNA species containing a 5' guanosine.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Deleção de Genes , Genes de Helmintos , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Homologia de Sequência de AminoácidosRESUMO
In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.
Assuntos
Instabilidade Genômica , Neoplasias , Humanos , Reparo do DNA/genética , Reparo do DNA por Junção de Extremidades , Neoplasias/genética , Replicação do DNA , Aberrações CromossômicasRESUMO
BACKGROUND: The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at developmental gene promoters and imprinted gene loci. This unique chromatin packaging at certain gene promoters provides these genomic loci the ability to convey instructive epigenetic information to the zygote, potentially expanding the role and significance of the sperm epigenome in embryogenesis. We hypothesize that changes in chromatin packaging may be associated with poor reproductive outcome. METHODS: Seven patients with reproductive dysfunction were recruited: three had unexplained poor embryogenesis during IVF and four were diagnosed with male infertility and previously shown to have altered protamination. Genome-wide analysis of the location of histones and histone modifications was analyzed by isolation and purification of DNA bound to histones and protamines. The histone-bound fraction of DNA was analyzed using high-throughput sequencing, both initially and following chromatin immunoprecipitation. The protamine-bound fraction was hybridized to agilent arrays. DNA methylation was examined using bisulfite sequencing. RESULTS: Unlike fertile men, five of seven infertile men had non-programmatic (randomly distributed) histone retention genome-wide. Interestingly, in contrast to the total histone pool, the localization of H3 Lysine 4 methylation (H3K4me) or H3 Lysine 27 methylation (H3K27me) was highly similar in the gametes of infertile men compared with fertile men. However, there was a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes. Finally, the methylation status of candidate developmental promoters and imprinted loci were altered in a subset of the infertile men. CONCLUSIONS: This initial genome-wide analysis of epigenetic markings in the sperm of infertile men demonstrates differences in composition and epigenetic markings compared with fertile men, especially at certain imprinted and developmental loci. Although no single locus displays a complete change in chromatin packaging or DNA modification, the data suggest that moderate changes throughout the genome exist and may have a cumulative detrimental effect on fecundity.
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Epigênese Genética , Genes Controladores do Desenvolvimento , Genoma Humano , Impressão Genômica , Infertilidade Masculina/genética , Espermatozoides/metabolismo , Adulto , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Epigenômica , Fertilização in vitro , Histonas , Humanos , Masculino , Nucleossomos/metabolismo , Regiões Promotoras GenéticasRESUMO
The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ2=5.4, P = 0.021). Guiding ctDNA detection in pre-operative ccfDNA based on mutations present in tumor DNA yielded a similar survival analysis. Detection of ctDNA in the post-operative ccfDNA with or without tumor-informed guidance was not associated with outcomes. Therefore, the detection of PDAC-derived ctDNA during a broad and untargeted survey of ccfDNA with NGS may be a valuable, non-invasive, prognostic biomarker to integrate into the clinical assessment and management of patients prior to surgery.
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Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pancreáticas/genética , Análise de Sequência de DNA/métodos , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/diagnóstico , DNA Tumoral Circulante/sangue , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , PrognósticoRESUMO
BACKGROUND: With the rapidly falling cost and availability of high throughput sequencing and microarray technologies, the bottleneck for effectively using genomic analysis in the laboratory and clinic is shifting to one of effectively managing, analyzing, and sharing genomic data. RESULTS: Here we present three open-source, platform independent, software tools for generating, analyzing, distributing, and visualizing genomic data. These include a next generation sequencing/microarray LIMS and analysis project center (GNomEx); an application for annotating and programmatically distributing genomic data using the community vetted DAS/2 data exchange protocol (GenoPub); and a standalone Java Swing application (GWrap) that makes cutting edge command line analysis tools available to those who prefer graphical user interfaces. Both GNomEx and GenoPub use the rich client Flex/Flash web browser interface to interact with Java classes and a relational database on a remote server. Both employ a public-private user-group security model enabling controlled distribution of patient and unpublished data alongside public resources. As such, they function as genomic data repositories that can be accessed manually or programmatically through DAS/2-enabled client applications such as the Integrated Genome Browser. CONCLUSIONS: These tools have gained wide use in our core facilities, research laboratories and clinics and are freely available for non-profit use. See http://sourceforge.net/projects/gnomex/, http://sourceforge.net/projects/genoviz/, and http://sourceforge.net/projects/useq.
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Genoma , Genômica/métodos , Software , Gráficos por Computador , Bases de Dados Factuais , Internet , Interface Usuário-ComputadorRESUMO
Challenges with distinguishing circulating tumor DNA (ctDNA) from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Here we show early and random PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding, removal of artifacts due to clonal hematopoiesis of indeterminate potential, and suppression of patterned errors. We also demonstrate sample duplicates are necessary to eliminate the stochastic noise associated with NGS. Integration of sample duplicates into NGS analytics may broaden ctDNA applications by removing NGS-related errors that confound identification of true very low frequency variants during searches for ctDNA without a priori knowledge of specific mutations to target.
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DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Código de Barras de DNA Taxonômico , Feminino , Hematopoese/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
HSUR2 is a viral non-coding RNA (ncRNA) that functions as a microRNA (miRNA) adaptor. HSUR2 inhibits apoptosis in infected cells by recruiting host miRNAs miR-142-3p and miR-16 to mRNAs encoding apoptotic factors. HSUR2's target recognition mechanism is not understood. It is also unknown why HSUR2 utilizes miR-16 to downregulate only a subset of transcripts. We developed a general method for individual-nucleotide resolution RNA-RNA interaction identification by crosslinking and capture (iRICC) to identify sequences mediating interactions between HSUR2 and target mRNAs in vivo. Mutational analyses confirmed identified HSUR2-mRNA interactions and validated iRICC as a method that confidently determines sequences mediating RNA-RNA interactions in vivo. We show that HSUR2 does not display a 'seed' region to base-pair with most target mRNAs, but instead uses different regions to interact with different transcripts. We further demonstrate that this versatile mode of interaction via variable base-pairing provides HSUR2 with a mechanism for differential miRNA recruitment.
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Pareamento de Bases , Herpesvirus Saimiriíneo 2/genética , Interações Hospedeiro-Patógeno , MicroRNAs/genética , RNA Mensageiro/genética , RNA Viral/genética , Animais , Linhagem Celular , Análise Mutacional de DNA , Herpesvirus Saimiriíneo 2/crescimento & desenvolvimento , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismoRESUMO
BACKGROUND: High throughput signature sequencing holds many promises, one of which is the ready identification of in vivo transcription factor binding sites, histone modifications, changes in chromatin structure and patterns of DNA methylation across entire genomes. In these experiments, chromatin immunoprecipitation is used to enrich for particular DNA sequences of interest and signature sequencing is used to map the regions to the genome (ChIP-Seq). Elucidation of these sites of DNA-protein binding/modification are proving instrumental in reconstructing networks of gene regulation and chromatin remodelling that direct development, response to cellular perturbation, and neoplastic transformation. RESULTS: Here we present a package of algorithms and software that makes use of control input data to reduce false positives and estimate confidence in ChIP-Seq peaks. Several different methods were compared using two simulated spike-in datasets. Use of control input data and a normalized difference score were found to more than double the recovery of ChIP-Seq peaks at a 5% false discovery rate (FDR). Moreover, both a binomial p-value/q-value and an empirical FDR were found to predict the true FDR within 2-3 fold and are more reliable estimators of confidence than a global Poisson p-value. These methods were then used to reanalyze Johnson et al.'s neuron-restrictive silencer factor (NRSF) ChIP-Seq data without relying on extensive qPCR validated NRSF sites and the presence of NRSF binding motifs for setting thresholds. CONCLUSION: The methods developed and tested here show considerable promise for reducing false positives and estimating confidence in ChIP-Seq data without any prior knowledge of the chIP target. They are part of a larger open source package freely available from http://useq.sourceforge.net/.
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Imunoprecipitação da Cromatina/métodos , Algoritmos , Animais , Transformação Celular Neoplásica , Cromatina/química , Reações Falso-Positivas , Regulação da Expressão Gênica , Genoma , Histonas/química , Humanos , Neurônios/metabolismo , Distribuição de Poisson , Reação em Cadeia da Polimerase , Proteínas/química , Análise de Sequência de DNARESUMO
Circulating tumor-derived cell-free DNA (ctDNA) enables non-invasive diagnosis, monitoring, and treatment susceptibility testing in human cancers. However, accurate detection of variant alleles, particularly during untargeted searches, remains a principal obstacle to widespread application of cell-free DNA in clinical oncology. In this study, isolation of short cell-free DNA fragments is shown to enrich for tumor variants and improve correction of PCR- and sequencing-associated errors. Subfractions of the mononucleosome of circulating cell-free DNA (ccfDNA) were isolated from patients with melanoma, pancreatic ductal adenocarcinoma, and colorectal adenocarcinoma using a high-throughput-capable automated gel-extraction platform. Using a 128-gene (128 kb) custom next-generation sequencing panel, variant alleles were on average 2-fold enriched in the short fraction (median insert size: ~142 bp) compared to the original ccfDNA sample, while 0.7-fold reduced in the fraction corresponding to the principal peak of the mononucleosome (median insert size: ~167 bp). Size-selected short fractions compared to the original ccfDNA yielded significantly larger family sizes (i.e., PCR duplicates) during in silico consensus sequence interpretation via unique molecular identifiers. Increments in family size were associated with a progressive reduction of PCR and sequencing errors. Although consensus read depth also decreased at larger family sizes, the variant allele frequency in the short ccfDNA fraction remained consistent, while variant detection in the original ccfDNA was commonly lost at family sizes necessary to minimize errors. These collective findings support the automated extraction of short ccfDNA fragments to enrich for ctDNA while concomitantly reducing false positives through in silico error correction.