CalcBiomin

This repository contains scripts and input data for the analyses described in the manuscript:
“Genetic parallels in biomineralization of the calcareous sponge Sycon ciliatum and stony corals”.

The repository is organized to replicate RNA-seq analyses (DESeq2 DGE, WGCNA), annotation, and GO enrichment.

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Repository structure

inputfiles/                     # Input data used in the analyses
    peptides_spicules/          # Peptide annotation data for spicule proteins
    annotation_files/           # Gene/transcript annotation data
        petide_blast_and_annotation/
    count_data/                 # # Read count tables and metadata (see details below)
        counts_regeneration/
        gene_counts_combined/
        counts_body_parts/
        counts_info/
    GOterms/                    # GO terms obtained with the script perl/Get_GO_Annotations_v2.pl
        gene_GO_terms/
        transcript_GOterms/

R_scripts/                      # R scripts for statistical analyses
    DESeq2/                     # Differential gene expression with DESeq2
        plots_DESeq_body-parts/
        plots_DESeq_regeneration/
    GOs/                        # GO term enrichment analysis
    wcgna/                      # WGCNA co-expression analysis
        plots/

perl/                           # Perl scripts for preprocessing and annotation
transcriptome/                  # Transcriptome reference data

Count Matrices for DGE and WGCNA Analyses

Five specimens of Sycon ciliatum were dissected into three body parts:

1. Oscular region
2. Inner sponge wall
3. Outer sponge wall

RNA-seq was conducted for each body part, and the raw reads were mapped against a Sycon transcriptome. Mapped reads were further filtered to remove sequences from commensal organisms. Gene and transcript counts for each filtered set were obtained with SALMON and combined into count matrices for the body parts experiment and for the regeneration experiment.

• Count matrices for the body parts experiment are in:
  inputfiles/count_data/counts_body_parts/

• For the regeneration experiment, raw reads (PRJNA628727) were processed in the same way. Count matrices are in:
  inputfiles/count_data/counts_regeneration/

• A matrix combining the gene counts of both experiments is in:
  inputfiles/count_data/gene_counts_combined/

• Sample information for these matrices is in:
  inputfiles/count_data/counts_info/

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Requirements

• R (≥ 4.0)
• R packages commonly used across scripts:
  DESeq2, genefilter, WGCNA, topGO, ggplot2, reshape2, svglite
  (Some scripts may require additional packages; see comments within each script.)
• Perl (for scripts in the perl/ directory)

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Input files

All required input files are included under inputfiles/, including:

• Count data tables (gene/transcript level) and sample metadata
• Gene/transcript annotation and BLAST results
• GO term mapping files

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How to run

1. Open the relevant script from R_scripts/ or perl/.

2. Follow the comments in each script regarding working directories and paths
   (several scripts set their own working directory and use repo-relative paths).

3. Run from R or the command line, e.g.:

   Rscript R_scripts/<subfolder>/<script_name>.R

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Outputs

Depending on the script:

• Differential expression result tables (CSV)
• Plots (SVG/PDF) saved in subfolders such as R_scripts/DESeq2/plots_* or R_scripts/wcgna/plots
• GO enrichment result tables
• WGCNA module gene lists

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