RESUMO
The concept of a nucleic acid barcode applied to pathogen genomes is easy to grasp and the many possible uses are straightforward. But implementation may not be easy, especially when growing through multiple generations or assaying the pathogen long-term. The potential problems include: the barcode might alter fitness, the barcode may accumulate mutations, and construction of the marked pathogens may result in unintended barcodes that are not as designed. Here, we generate approximately 5,000 randomized barcodes in the genome of the prototypic small DNA virus murine polyomavirus. We describe the challenges faced with interpreting the barcode sequences obtained from the library. Our Illumina NextSeq sequencing recalled much greater variation in barcode sequencing reads than the expected 5,000 barcodes-necessarily stemming from the Illumina library processing and sequencing error. Using data from defined control virus genomes cloned into plasmid backbones we develop a vetted post-sequencing method to cluster the erroneous reads around the true virus genome barcodes. These findings may foreshadow problems with randomized barcodes in other microbial systems and provide a useful approach for future work utilizing nucleic acid barcoded pathogens.
Assuntos
Vírus de DNA , Ácidos Nucleicos , Camundongos , Animais , Vírus de DNA/genéticaRESUMO
Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling-RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined.
RESUMO
Clostridium taeniosporum, a non-pathogenic anaerobe closely related to the C. botulinum Group II members, was isolated from Crimean lake silt about 60 years ago. Its endospores are surrounded by an encasement layer which forms a trunk at one spore pole to which about 12-14 large, ribbon-like appendages are attached. The genome consists of one 3,264,813 bp, circular chromosome (with 26.6% GC) and three plasmids. The chromosome contains 2,892 potential protein coding sequences: 2,124 have specific functions, 147 have general functions, 228 are conserved but without known function and 393 are hypothetical based on the fact that no statistically significant orthologs were found. The chromosome also contains 101 genes for stable RNAs, including 7 rRNA clusters. Over 84% of the protein coding sequences and 96% of the stable RNA coding regions are oriented in the same direction as replication. The three known appendage genes are located within a single cluster with five other genes, the protein products of which are closely related, in terms of sequence, to the known appendage proteins. The relatedness of the deduced protein products suggests that all or some of the closely related genes might code for minor appendage proteins or assembly factors. The appendage genes might be unique among the known clostridia; no statistically significant orthologs were found within other clostridial genomes for which sequence data are available. The C. taeniosporum chromosome contains two functional prophages, one Siphoviridae and one Myoviridae, and one defective prophage. Three plasmids of 5.9, 69.7 and 163.1 Kbp are present. These data are expected to contribute to future studies of developmental, structural and evolutionary biology and to potential industrial applications of this organism.
Assuntos
Clostridium/genética , Genoma Bacteriano , Esporos Bacterianos , Clostridium/metabolismo , Filogenia , Prófagos/classificação , Prófagos/genética , Origem de Replicação , Selenoproteínas/metabolismoRESUMO
Gestational exposures to estrogenic compounds, both endogenous hormones and exogenous endocrine-disrupting chemicals (EDCs), have long-term effects on reproductive physiology and behavior. We tested the hypothesis that prenatal treatment of rats with low doses of Aroclor 1221 (A1221), a weakly estrogenic polychlorinated biphenyl mix previously used in industry, or estradiol benzoate (EB), alters development of the hypothalamus in a sexually dimorphic manner and subsequently perturbs reproductive function. Pregnant Sprague-Dawley rats were injected on embryonic days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or EB (50 µg/kg). Developmental milestones were monitored, and on postnatal days 15, 30, 45, and 90, 1 male and 1 female per litter were euthanized. Because of their key roles in the mediation of steroid actions on reproductive function, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) were punched for a low-density quantitative PCR array of 48 neuroendocrine genes and analysis of DNA methylation of a subset of genes. Gestational exposure to A1221 or EB delayed the timing of puberty in males and disrupted estrous cyclicity in females. In the AVPV, 28 genes were affected by treatment in a developmental stage-specific manner, mostly in females, which exhibited a masculinized expression profile. This included 2 clock genes, Per2 and Arntl, implicating circadian circuits as being vulnerable to endocrine disruption. DNA methylation analysis of 2 genes, Per2 and Ar, showed no effect of EDCs and suggested alternative mechanisms for the altered mRNA levels. In the ARC, 12 genes were affected by treatment, mostly in males, again with dynamic developmental changes. Bionetwork analysis of relationships among genes, hormones, and physiological markers showed sexually dimorphic effects of estrogenic EDC exposures, with the female AVPV and the male ARC being most vulnerable, and provided novel relationships among hypothalamic genes and postnatal reproductive maturation.