RESUMEN
OBJECTIVES: Measurable residual disease flow cytometry (MRD-FC) and molecular studies are the most sensitive methods for detecting residual malignant populations after therapy for TP53-mutated acute myeloid leukemia and myelodysplastic neoplasms (TP53+ AML/MDS). However, their sensitivity is limited in suboptimal aspirates or when the immunophenotype of the neoplastic blasts overlaps with erythroids or normal maturing myeloid cells. In this study, we set out to determine if p53 immunohistochemistry (IHC) correlates with MRD-FC and next-generation sequencing (NGS) in the posttherapy setting and to determine the utility of p53 IHC to detect residual disease in the setting of negative or equivocal MRD-FC. METHODS: We retrospectively identified 28 pre- and posttherapy bone marrow biopsy specimens from 9 patients with TP53+ AML/MDS and a p53 overexpressor phenotype by IHC (strong 3+ staining at initial diagnosis). Next-generation sequencing and/or MRD-FC results were collected for each specimen. RESULTS: Using a threshold of more than ten 2-3+ cells in any one 400× field, p53 IHC detected residual disease with a sensitivity of 94% and a specificity of 89%. The threshold used in this study showed a high degree of concordance among 6 blinded pathologists (Fleiss κ = 0.97). CONCLUSIONS: Our study suggests that p53 IHC can be used as a rapid tool (within 24 hours) to aid in the detection of residual disease that may complement MRD-FC or NGS in cases in which the flow cytometry immunophenotype is equivocal and/or the bone marrow aspirate is suboptimal.
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Inmunohistoquímica , Leucemia Mieloide Aguda , Mutación , Síndromes Mielodisplásicos , Neoplasia Residual , Proteína p53 Supresora de Tumor , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/patología , Síndromes Mielodisplásicos/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Estudios Retrospectivos , Masculino , Persona de Mediana Edad , Femenino , Anciano , Secuenciación de Nucleótidos de Alto Rendimiento , Citometría de Flujo , Anciano de 80 o más Años , Adulto , Inmunofenotipificación , Sensibilidad y Especificidad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisisRESUMEN
OBJECTIVES: Gene rearrangements frequently act as oncogenic driver mutations and determine the onset and progression of cancer. RNA-based next-generation sequencing (NGS) is being used with increasing frequency for solid tumors. The purpose of our study is to investigate the feasibility and utility of an RNA-based NGS fusion panel for solid tumors. METHODS: We conducted a retrospective, single-institution review of fusion panels requested between May 2022 and March 2023. Demographic, clinical, pathologic, and molecular findings of the patients were reviewed. The utility of the RNA-based NGS fusion panel for the pathologic diagnosis of solid tumors was assessed. RESULTS: Our study included 345 cases, and a fusion event was identified in 24.3% (78/321) of cases. Among the 110 cases submitted for diagnostic purposes, a fusion event was detected in 42.7% (47/110) of cases. The results led to refinement or clarification of the initial diagnosis in 31.9% (15/47) of cases and agreement or support for the initial diagnosis in 59.6% (28/47) of cases. Furthermore, our study indicated that the overall cellularity (tumor and normal tissue) of the tested specimen influences the success of the testing process. CONCLUSIONS: In summary, this study demonstrated the feasibility and utility of an RNA-based NGS fusion panel for a wide variety of solid tumors in the appropriate clinicopathologic context. These findings warrant further validation in larger studies involving multiple institutional patient cohorts.
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Neoplasias , ARN , Humanos , Estudios Retrospectivos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patología , Reordenamiento Génico , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies. A custom bioinformatics pipeline and database was also developed to facilitate minimal/measurable residual disease analysis and clinical reporting. This assay demonstrated excellent test performance characteristics, achieving a sensitivity of 1 of 100,000 T-cell equivalents for the DNA inputs evaluated and high concordance with orthogonal testing methods. This assay was further utilized to correlate disease burden in several patients, demonstrating its potential utility for monitoring patients with T-cell malignancies.
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Linfoma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linfocitos T , Receptores de Antígenos de Linfocitos T/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genéticaRESUMEN
CONTEXT.: Mutational signatures have been described in the literature and a few centers have implemented pipelines for clinical reporting. OBJECTIVE.: To describe the performance of a mutational signature caller with clinical samples sequenced on a targeted next-generation sequencing panel with a small genomic footprint. DESIGN.: One thousand six hundred eighty-two clinical samples were analyzed for the presence of mutational signatures using deconstructSigs on variant calls with at least 20 variant reads. RESULTS.: Signature 10 (associated with POLe mutation) achieved separation of cases and controls in hypermutated samples. Signatures 4 (associated with tobacco smoking) and 7 (associated with ultraviolet radiation) as an indicator of pulmonary or cutaneous primary sites showed moderate sensitivity and high specificity at optimal cutpoints. Mutational signatures in malignancies with unknown primaries were somewhat consistent with the clinically suspected primary site, with an apparent dose-response relationship between the number of variants analyzed and the ability of mutational signature analysis to correctly suggest a primary site. CONCLUSIONS.: Mutational signatures represent an opportunity for orthogonal testing of primary site, which may be particularly useful in supporting cutaneous or pulmonary sites in poorly differentiated neoplasms. Tobacco smoking, ultraviolet radiation, and POLe mutational signatures are the most appropriate signatures for implementation. Even relatively small numbers of variants appear capable of supporting a clinically suspected primary.
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Biomarcadores de Tumor/genética , Análisis Mutacional de ADN , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Pulmonares/genética , Mutación , Neoplasias Inducidas por Radiación/genética , Neoplasias Cutáneas/genética , Estudios de Casos y Controles , ADN Polimerasa II/genética , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Valor Predictivo de las Pruebas , Medición de Riesgo , Factores de Riesgo , Fumar Tabaco/efectos adversos , Fumar Tabaco/genética , Rayos Ultravioleta/efectos adversosRESUMEN
PURPOSE: Improved second-tier tools are needed to reduce false-positive outcomes in newborn screening (NBS) for inborn metabolic disorders on the Recommended Universal Screening Panel (RUSP). METHODS: We designed an assay for multiplex sequencing of 72 metabolic genes (RUSPseq) from newborn dried blood spots. Analytical and clinical performance was evaluated in 60 screen-positive newborns for methylmalonic acidemia (MMA) reported by the California Department of Public Health NBS program. Additionally, we trained a Random Forest machine learning classifier on NBS data to improve prediction of true and false-positive MMA cases. RESULTS: Of 28 MMA patients sequenced, we found two pathogenic or likely pathogenic (P/LP) variants in a MMA-related gene in 24 patients, and one pathogenic variant and a variant of unknown significance (VUS) in 1 patient. No such variant combinations were detected in MMA false positives and healthy controls. Random Forest-based analysis of the entire NBS metabolic profile correctly identified the MMA patients and reduced MMA false-positive cases by 51%. MMA screen-positive newborns were more likely of Hispanic ethnicity. CONCLUSION: Our two-pronged approach reduced false positives by half and provided a reportable molecular finding for 89% of MMA patients. Challenges remain in newborn metabolic screening and DNA variant interpretation in diverse multiethnic populations.
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Errores Innatos del Metabolismo de los Aminoácidos/sangre , Variación Genética , Errores Innatos del Metabolismo/sangre , Tamizaje Neonatal , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/patología , Pruebas con Sangre Seca , Femenino , Humanos , Recién Nacido , Aprendizaje Automático , Masculino , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/patologíaRESUMEN
BACKGROUND: Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation. METHODS: We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients. RESULTS: For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR. CONCLUSIONS: Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.
RESUMEN
Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Pruebas con Sangre Seca/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Tamizaje Neonatal/métodos , Costos y Análisis de Costo , Fibrosis Quística/diagnóstico , Variaciones en el Número de Copia de ADN , Cartilla de ADN , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Humanos , Recién Nacido , Reacción en Cadena de la Polimerasa Multiplex/economía , Reacción en Cadena de la Polimerasa Multiplex/métodos , Tamizaje Neonatal/economía , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
RATIONALE: Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming. OBJECTIVE: We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost. METHODS AND RESULTS: We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample. CONCLUSIONS: Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.
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Análisis Costo-Beneficio , Cardiopatías/economía , Cardiopatías/genética , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuencia de Bases , Marcación de Gen/economía , Marcación de Gen/métodos , Cardiopatías/diagnóstico , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , Factores de TiempoRESUMEN
The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.
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Bacterias/aislamiento & purificación , Sondas Moleculares/química , Bacterias/clasificación , Bacterias/genética , Técnicas Bacteriológicas/métodos , ADN Bacteriano/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/síntesis química , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
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Células/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Resistencia a Medicamentos/genética , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular Tumoral , Haploinsuficiencia , Humanos , Farmacogenética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
Reproductive tract infection is a major initiator of preterm birth (PTB). The objective of this prospective cohort study of 88 participants was to determine whether PTB correlates with the vaginal microbiome during pregnancy. Total DNA was purified from posterior vaginal fornix swabs during gestation. The 16S ribosomal RNA gene was amplified using polymerase chain reaction primers, followed by chain-termination sequencing. Bacteria were identified by comparing contig consensus sequences with the Ribosomal Database Project. Dichotomous responses were summarized via proportions and continuous variables via means ± standard deviation. Mean Shannon Diversity index differed by Welch t test (P = .00016) between caucasians with PTB and term gestation. Species diversity was greatest among African Americans (P = .0045). Change in microbiome/Lactobacillus content and presence of putative novel/noxious bacteria did not correlate with PTB. We conclude that uncultured vaginal bacteria play an important role in PTB and race/ethnicity and sampling location are important determinants of the vaginal microbiome.
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Bacterias/clasificación , Recien Nacido Prematuro , Microbiota , Nacimiento Prematuro/microbiología , Vagina/microbiología , Adulto , Negro o Afroamericano , Asiático , Bacterias/genética , Bacterias/aislamiento & purificación , Estudios de Casos y Controles , ADN Bacteriano/aislamiento & purificación , Femenino , Hispánicos o Latinos , Humanos , Lactobacillus/clasificación , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Metagenoma , Metagenómica , Embarazo , Nacimiento Prematuro/etnología , Estudios Prospectivos , ARN Ribosómico 16S/genética , Ribotipificación , Factores de Riesgo , San Francisco/epidemiología , Población BlancaRESUMEN
The automated cell, compound and environment screening system (ACCESS) was developed as an automated platform for chemogenomic research. In the yeast Saccharomyces cerevisiae, a number of genomic screens rely on the modulation of gene dose to determine the mode of action of bioactive compounds or the effects of environmental/compound perturbations. These and other phenotypic experiments have been shown to benefit from high-resolution growth curves and a highly automated controlled environment system that enables a wide range of multi-well assays that can be run over many days without any manual intervention. Furthermore, precise control of drug dosing, timing of drug exposure, and precise timing of cell harvesting at specific generation times are important for optimal results. Some of these benefits include the ability to derive fine distinctions between growth rates of mutant strains (1) and the discovery of novel compounds and drug targets (2). The automation has also enabled large-scale screening projects with over 100,000 unique compounds screened to date including a thousand genome-wide screens (3). The ACCESS system also has a diverse set of software tools to enable users to set up, run, annotate, and evaluate complex screens with minimal training.
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Ambiente , Genómica/métodos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Automatización , Gráficos por Computador , Bases de Datos Factuales , Evaluación Preclínica de Medicamentos , Genómica/instrumentación , Laboratorios , Robótica , Saccharomyces cerevisiae/efectos de los fármacos , Programas Informáticos , Estadística como Asunto , Interfaz Usuario-ComputadorRESUMEN
Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.
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Candida albicans/genética , Elementos Transponibles de ADN , Descubrimiento de Drogas/métodos , Anotación de Secuencia Molecular/métodos , Terapia Molecular Dirigida/métodos , Organismos Modificados Genéticamente , Candida albicans/fisiología , Elementos Transponibles de ADN/genética , Alimentos , Perfilación de la Expresión Génica , Biblioteca Genómica , Haploinsuficiencia/genética , Análisis por Micromatrices , Modelos Biológicos , Mutagénesis Insercional/métodos , Organismos Modificados Genéticamente/genética , Lugares Marcados de SecuenciaRESUMEN
Systems-level analyses of non-model microorganisms are limited by the existence of numerous uncharacterized genes and a corresponding over-reliance on automated computational annotations. One solution to this challenge is to disrupt gene function using DNA tag technology, which has been highly successful in parallelizing reverse genetics in Saccharomyces cerevisiae and has led to discoveries in gene function, genetic interactions and drug mechanism of action. To extend the yeast DNA tag methodology to a wide variety of microorganisms and applications, we have created a universal, sequence-verified TagModule collection. A hallmark of the 4280 TagModules is that they are cloned into a Gateway entry vector, thus facilitating rapid transfer to any compatible genetic system. Here, we describe the application of the TagModules to rapidly generate tagged mutants by transposon mutagenesis in the metal-reducing bacterium Shewanella oneidensis MR-1 and the pathogenic yeast Candida albicans. Our results demonstrate the optimal hybridization properties of the TagModule collection, the flexibility in applying the strategy to diverse microorganisms and the biological insights that can be gained from fitness profiling tagged mutant collections. The publicly available TagModule collection is a platform-independent resource for the functional genomics of a wide range of microbial systems in the post-genome era.
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Candida albicans/genética , Mutagénesis Insercional/métodos , Shewanella/genética , Elementos Transponibles de ADN , Análisis de Secuencia por Matrices de Oligonucleótidos , Lugares Marcados de SecuenciaRESUMEN
Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.
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Diseño de Fármacos , Genoma Fúngico , Genómica/métodos , Compuestos Orgánicos/farmacología , Genes Fúngicos/efectos de los fármacos , Compuestos Orgánicos/síntesis química , Relación Estructura-Actividad , LevadurasRESUMEN
Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes ( approximately 80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.
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Genes Esenciales , Genes Fúngicos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Medios de Cultivo , Farmacorresistencia Fúngica Múltiple , Eliminación de Gen , Genes MDR , Genómica , Heterocigoto , Homocigoto , Redes y Vías Metabólicas/efectos de los fármacos , Familia de Multigenes , Fenotipo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single- and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination-derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.
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ARN Helicasas DEAD-box/genética , Eliminación de Gen , Genes Fúngicos , Metilmetanosulfonato/toxicidad , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Reparación del ADN , Modelos Genéticos , Datos de Secuencia Molecular , RecQ Helicasas/genéticaRESUMEN
Molecular barcode arrays allow the analysis of thousands of biological samples in parallel through the use of unique 20-base-pair (bp) DNA tags. Here we present a new barcode array, which is unique among microarrays in that it includes at least five replicates of every tag feature. The use of smaller dispersed replicate features dramatically improves performance versus a single larger feature and allows the correction of previously undetectable hybridization defects.
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Sondas de ADN/genética , Etiquetas de Secuencia Expresada , Hibridación Fluorescente in Situ/instrumentación , Técnicas de Sonda Molecular/instrumentación , Alineación de Secuencia/instrumentación , Análisis de Secuencia de ADN/instrumentación , Secuencia de Bases , Diseño de Equipo , Análisis de Falla de Equipo , Hibridación Fluorescente in Situ/métodos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.
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Cryptococcus neoformans/genética , Genoma Fúngico , Empalme Alternativo , Pared Celular/metabolismo , Cromosomas Fúngicos/genética , Biología Computacional , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/fisiología , Elementos Transponibles de ADN , Proteínas Fúngicas/metabolismo , Biblioteca de Genes , Genes Fúngicos , Humanos , Intrones , Datos de Secuencia Molecular , Fenotipo , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Polisacáridos/metabolismo , ARN sin Sentido , Análisis de Secuencia de ADN , Transcripción Genética , Virulencia , Factores de Virulencia/metabolismoRESUMEN
To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.