RESUMO
MOTIVATION: The diverse functionalities of RNA can be attributed to its capacity to form complex and varied structures. The recent proliferation of new structure probing techniques coupled with high-throughput sequencing has helped RNA studies expand in both scope and depth. Despite differences in techniques, most experiments face similar challenges in reproducibility due to the stochastic nature of chemical probing and sequencing. As these protocols expand to transcriptome-wide studies, quality control becomes a more daunting task. General and efficient methodologies are needed to quantify variability and quality in the wide range of current and emerging structure probing experiments. RESULTS: We develop metrics to rapidly and quantitatively evaluate data quality from structure probing experiments, demonstrating their efficacy on both small synthetic libraries and transcriptome-wide datasets. We use a signal-to-noise ratio concept to evaluate replicate agreement, which has the capacity to identify high-quality data. We also consider and compare two methods to assess variability inherent in probing experiments, which we then utilize to evaluate the coverage adjustments needed to meet desired quality. The developed metrics and tools will be useful in summarizing large-scale datasets and will help standardize quality control in the field. AVAILABILITY AND IMPLEMENTATION: The data and methods used in this article are freely available at: http://bme.ucdavis.edu/aviranlab/SPEQC_software CONTACT: saviran@ucdavis.eduSupplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional/métodos , RNA/química , Análise de Sequência de RNA/métodos , Modelos Estatísticos , Controle de Qualidade , Reprodutibilidade dos Testes , Análise de Sequência de RNA/normas , Razão Sinal-RuídoRESUMO
The formation of reiterated somites along the vertebrate body axis is controlled by the segmentation clock, a molecular oscillator expressed within presomitic mesoderm (PSM) cells. Although PSM cells oscillate autonomously, they coordinate with neighboring cells to generate a sweeping wave of cyclic gene expression through the PSM that has a periodicity equal to that of somite formation. The velocity of each wave slows as it moves anteriorly through the PSM, although the dynamics of clock slowing have not been well characterized. Here, we investigate segmentation clock dynamics in the anterior PSM in developing zebrafish embryos using an in vivo clock reporter, her1:her1-venus. The her1:her1-venus reporter has single-cell resolution, allowing us to follow segmentation clock oscillations in individual cells in real-time. By retrospectively tracking oscillations of future somite boundary cells, we find that clock reporter signal increases in anterior PSM cells and that the periodicity of reporter oscillations slows to about â¼1.5 times the periodicity in posterior PSM cells. This gradual slowing of the clock in the anterior PSM creates peaks of clock expression that are separated at a two-segment periodicity both spatially and temporally, a phenomenon we observe in single cells and in tissue-wide analyses. These results differ from previous predictions that clock oscillations stop or are stabilized in the anterior PSM. Instead, PSM cells oscillate until they incorporate into somites. Our findings suggest that the segmentation clock may signal somite formation using a phase gradient with a two-somite periodicity.
Assuntos
Embrião não Mamífero/metabolismo , Peixe-Zebra/embriologia , Animais , Estudos Retrospectivos , Somitos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Vertebrate body segmentation is controlled by the segmentation clock, a molecular oscillator involving transcriptional oscillations of cyclic genes in presomitic mesoderm cells. The rapid and highly dynamic nature of this oscillating system has proved challenging for study at the single-cell level. We achieved visualization of clock activity with a cellular level of resolution in living embryos, allowing direct comparison of oscillations in neighbor cells. We provide direct evidence that presomitic mesoderm cells oscillate asynchronously in zebrafish Notch pathway mutants. By tracking oscillations in mitotic cells, we reveal that a robust cell-autonomous, Notch-independent mechanism resumes oscillations after mitosis. Finally, we find that cells preferentially divide at a certain oscillation phase, likely reducing the noise generated by cell division in cell synchrony and suggesting an intriguing relationship between the mitotic cycle and clock oscillation.