Gal4-sfGFP-Fis1 variant amplicon sequencing
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PROVIDER: PRJEB13011 | ENA |
REPOSITORIES: ENA
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Project description:Mediator (MED) is a conserved factor with important roles in basal and activated transcription. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. Rapid Tail depletion surprisingly reduces transcription from only a small subset of genes. At most of these Tail-dependent genes, in unperturbed conditions, MED is detected at both the UASs and promoters. In contrast, at most Tail-independent genes, we find MED primarily at promoters but not at the UASs. These results suggest that MED Tail and activator-mediated MED recruitment regulate only a small subset of genes. Further, we define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID and BET factors Bdf1/2. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.
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Project description:Mediator (MED) is a conserved factor with important roles in basal and activated transcription. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. Rapid Tail depletion surprisingly reduces transcription from only a small subset of genes. At most of these Tail-dependent genes, in unperturbed conditions, MED is detected at both the UASs and promoters. In contrast, at most Tail-independent genes, we find MED primarily at promoters but not at the UASs. These results suggest that MED Tail and activator-mediated MED recruitment regulate only a small subset of genes. Further, we define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID and BET factors Bdf1/2. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.
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Project description:BackgroundHaloplex targeted resequencing is a popular method to analyze both germline and somatic variants in gene panels. However, involved wet-lab procedures may introduce false positives that need to be considered in subsequent data-analysis. No variant filtering rationale addressing amplicon enrichment related systematic errors, in the form of an all-in-one package, exists to our knowledge.ResultsWe present pyAmpli, a platform independent parallelized Python package that implements an amplicon-based germline and somatic variant filtering strategy for Haloplex data. pyAmpli can filter variants for systematic errors by user pre-defined criteria. We show that pyAmpli significantly increases specificity, without reducing sensitivity, essential for reporting true positive clinical relevant mutations in gene panel data.ConclusionspyAmpli is an easy-to-use software tool which increases the true positive variant call rate in targeted resequencing data. It specifically reduces errors related to PCR-based enrichment of targeted regions.
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