Repo assembled by AATS and MvdD from own work & previous work by MWMM & SHC over the past few years.
Code is provided as is as supporting material. R version was 4.2.1 (2022-06-23) unless otherwise indicated.
Main sections:
- GRH-1 ChIP-seq processing
- promoter occupancy (RNA-polII ChIP-seq & mRNA-seq timecourse)
- phase enrichment analysis amongst putative GRH-1 targets
- segmentation model for image analysis
This folder contains RStudio notebooks that process the ChIP-sequencing data to called peaks, motifs and sites.
Processes WS220 exon definitions for coding transcripts & miRNA non-mitochondrial genes into exon GenomicRanges, pan-isoform gene promotoer & body GenomicRanges, annotated with gene symbols and transcript names, for further use.
Align ChIP-sequencing SE50 reads from both experiments/runs (2287R and 2633R) to the ce10 genome.
The ce10 genome was split into 500bp tiles. Tiles were qualified by GC content and by number of reads mapping to them. Input and ChIP samples (and enrichments derived therefrom) were examined for evidence of GC bias or other issues.
Peaks were called using MACS2 for sample input/ChIP pairs from both experiments. Peaks overlapping "overmapped" tiles were removed from later analysis. Peaks were then checked for issues (GC content, low mappability, discrepancy between MACS2 and manually-calculated enrichments...).
The ce10 genome was masked to remove unmappable regions. The 10% of leaste enriched peaks were filtered out. HOMER was then run using various setups. Obtained motifs were assessed manually across setups to select candidate GRH-1 motifs for further analysis. Scrambled versions of candidates were generated. The mappable ce10 genome was scanned for sites corresponding to candidate and scrambled motifs. Agreement between peaks and sites was evaluated in various ways. A final candidate motif was retained for further analyses.
Binding site information was aggregated to the peak level, and peaks were assigned to genes based on promoter overlap, gene body overlap, or by lying directly upstream (with a distance cutoff).
This folder contains scripts analysing the timecourse experiment looking into gene promoter occupancy (via pol-II ChIP-seq) and mRNA expression (RNA-seq).
This script counts hits to 1kb regions centered on putative TSSs (ie that of the combined "gene body"), from samples of Run 899 aligned to ce10. Counts are reported per gene, library-size normalised (min lib size, lib size from project alignment stats), log2-transformed w/ a pseudocount of 8, filtered to "expressed" genes (mean>3), then quantile normalised. Only counts for the pull-down samples (ie "polII", ie not input) are kept, 22h-33h. These are measures of promoter occupancy by RNA pol-II. This script was run with an unknown older version of R.
This script counts hits to gene exons, from samples of RNA-seq run 1314 aligned to ce10. Counts are aggregated to gene-level by qCount, library-size normalised (mean lib size, lib size from summed hit counts), log2-transformed w/ a pseudocount of 8. This script was run with an unknown older version of R.
This script loads previously-prepared promoter occupancy and mRNA expression level data, calculates correlation between the 2, and describes each gene in terms of each dataset, said correlation and oscillatory status.
This script takes site-peak-gene assignments generated above, derives various definitions of "putative GRH-1 target" from this data (eg thresholding on site score, peak enrichment, etc), and investigates whether:
- there is evidence of enrichment in oscillatory genes amongst putative GRH-1 targets
- there is evidence of enrichment in particular phases amongst oscillatory putative GRH-1 targets
This CNN model is used to segment worms from brightfield images.
The model can then be called using the workflows from the publicly available repository:
ggrosshans-jobsystem-workflows.
Instructions given there can be followed for correct installation.
Note: the path written in the ./fetch_data_and_models.sh need to be updated to your local path where this repository is saved.