Project description:Monitoring microbial communities can aid in understanding the state of these habitats. Environmental DNA (eDNA) techniques provide efficient and comprehensive monitoring by capturing broader diversity. Besides structural profiling, eDNA methods allow the study of functional profiles, encompassing the genes within the microbial community. In this study, three methodologies were compared for functional profiling of microbial communities in estuarine and coastal sites in the Bay of Biscay. The methodologies included inference from 16S metabarcoding data using Tax4Fun, GeoChip microarrays, and shotgun metagenomics.
Project description:Comparison to RNA-Seq showed different strengths and weaknesses for different regimes of expression strength. At sufficient read-depth, both platforms seemed comparable for gene-level expression profiling. Comparison to RNA-Seq showed comparable accuracy and precision with microarrays for ERCC spike-ins at medium to high expression levels. At low expression levels, the array showed signal attenuation but better precision, while RNA-Seq maintained accuracy albeit with highly inflated variance. In summary, both platforms can be meaningfully applied in a similar range of expression strength. Assessment of (precision, accuracy, reproducibility, mutual information, titration order consistency, and known mixing ratio recovery) by measurement of known-ratio mixtures of two RNA reference samples. The array probes 776 complex genes representative of the AceView gene model annotation, as well as 92 ERCC spike-in controls.
Project description:A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on isobaric TMT reporter ion quantification.
Project description:Using an integrated analysis of chromatin remodeler binding in unperturbed cells and nucleosome displacement activity upon rapid remodeler depletion or degradation, we investigate the interplay between these enzymes and their functional consequences. We show that many promoters are acted upon by multiple remodelers that operate either cooperatively or in opposition to determine the precise location of the key transcription start site-associated +1 nucleosome. Functional assays indicate that +1 nucleosome positioning reflects a trade-off between maximizing RNA Polymerase II recruitment and minimizing transcription initiation at incorrect sites. Interestingly, in the absence of any remodeling activity +1 nucleosomes largely maintain their positions. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to productive transcription initiation.
Project description:We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells. We examine the effect of the interaction between BEAF and polybromo on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells.