Assuntos
Biomarcadores Tumorais , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Genômica , Linfoma não Hodgkin/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Genômica/métodos , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/metabolismo , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Adulto JovemRESUMO
As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95-99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.
Assuntos
Análise Mutacional de DNA/métodos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Variações do Número de Cópias de DNA , Frequência do Gene , Humanos , Neoplasias/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais/genética , Cinesinas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ret/genética , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Biópsia , Transformação Celular Neoplásica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinesinas/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Camundongos , Células NIH 3T3 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidoresRESUMO
Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.
Assuntos
Automação Laboratorial , Exoma , Biblioteca Gênica , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Controle de QualidadeRESUMO
We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.
Assuntos
Processamento Eletrônico de Dados , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Análise de Sequência de DNA/métodos , Algoritmos , Humanos , MicroesferasRESUMO
An anionic amphiphilic dendrimer is reported that possesses increased cytotoxicological potency against prokaryotic cells compared to eukaryotic cells. The half-maximal effective concentration (EC50) for the dendrimer against Bacillus subtilis, a Gram-positive bacterial strain, was measured to be 4.1 x 10(-5) M, while that against human umbilical vein endothelial cells (HUVEC) was more than 36x greater at a value of 1.5 x 10(-3) M. EC50 ratios for two commercial amphiphiles, sodium dodecyl sulfate (SDS) and Triton X-100, in addition to a similar synthesized dendritic structure were at most only 3.8x greater. Furthermore, the observed EC50 values appear to be correlated to the critical aggregation constant (CAC) in solution suggesting a mechanism of action for these anionic amphiphilic dendrimers related to their supramolecular structures.
Assuntos
Antibacterianos/farmacologia , Dendrímeros/farmacologia , Ânions/química , Ânions/farmacologia , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Dendrímeros/química , Células Endoteliais/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Octoxinol/farmacologia , Dodecilsulfato de Sódio/farmacologiaRESUMO
An iterative position-specific score matrix (PSSM)-based approach was used to predict sigma(28) promoters in 11 Shewanella genomes. The Shewanella Correlation Browser was used to distinguish true-positive predictions from false-positive predictions in Shewanella oneidensis MR-1 by generating a sigma(28)-regulated transcriptional network from transcriptional profiling data. This dual-pronged approach identified several genes that have sigma(28) promoters and that may be involved with motility or chemotaxis in Shewanella.
Assuntos
Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Regiões Promotoras Genéticas , Shewanella/genética , Fator sigma/metabolismo , Algoritmos , Sítios de Ligação , Biologia ComputacionalRESUMO
Many Microbe Microarrays Database (M3D) is designed to facilitate the analysis and visualization of expression data in compendia compiled from multiple laboratories. M3D contains over a thousand Affymetrix microarrays for Escherichia coli, Saccharomyces cerevisiae and Shewanella oneidensis. The expression data is uniformly normalized to make the data generated by different laboratories and researchers more comparable. To facilitate computational analyses, M3D provides raw data (CEL file) and normalized data downloads of each compendium. In addition, web-based construction, visualization and download of custom datasets are provided to facilitate efficient interrogation of the compendium for more focused analyses. The experimental condition metadata in M3D is human curated with each chemical and growth attribute stored as a structured and computable set of experimental features with consistent naming conventions and units. All versions of the normalized compendia constructed for each species are maintained and accessible in perpetuity to facilitate the future interpretation and comparison of results published on M3D data. M3D is accessible at http://m3d.bu.edu/.
Assuntos
Bases de Dados Genéticas , Escherichia coli/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Saccharomyces cerevisiae/genética , Shewanella/genética , Gráficos por Computador , Escherichia coli/metabolismo , Internet , Saccharomyces cerevisiae/metabolismo , Shewanella/metabolismo , Interface Usuário-ComputadorRESUMO
To identify pathways of carbon utilization in the metal-reducing marine bacterium Shewanella oneidensis MR-1, we assayed the expression of cells grown with various carbon sources using a high-density oligonucleotide Affymetrix microarray. Our expression profiles reveal genes and regulatory mechanisms which govern the sensing, import, and utilization of the nucleoside inosine, the chitin monomer N-acetylglucosamine, and a casein-derived mixture of amino acids. Our analysis suggests a prominent role for the pentose-phosphate and Entner-Doudoroff pathways in energy metabolism, and regulatory coupling between carbon catabolism and electron acceptor pathways. In sum, these results indicate that S. oneidensis possesses a broader capacity for carbon utilization than previously reported, a view with implications for optimizing its role in microbial fuel cell and bioremediative applications.
