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analysis/

R scripts that reproduce all figures and supplementary tables in the OpenSplice paper. Each script is self-contained: source it from RStudio or run it with Rscript from the repository root.

Prerequisites

All scripts source analysis/config.R at the top, which defines every input/output path. Open OpenSplice.Rproj in RStudio so that here() resolves paths from the project root automatically — no manual path editing needed.

Before running any analysis script, the master table must exist:

source("analysis/00_master_table_creation.R")

Script index

00 — Master table

Script Description
00_master_table_creation.R Joins PSI measurements with variant coordinates, computes per-variant statistics (delta-PSI, delta-LogitPSI, z-scores, FDR), assigns genomic regions, and writes Supplementary_Table4.tsv (= the master table used by all downstream scripts) and results/psi_per_variant/coverage_per_exon.tsv.

01 — Dataset QC and overview

Script Description
01.1_replicates_correlation_plots.R Replicate correlation (GGally pairs plots) for all mutagenesis and WT screening libraries.
01.2_other_dataset_comparison.R Comparison against external datasets: FAS INDEL vs. Baeza-Centurion et al. 2025, single-clone gel validation, and SpliceVarDB.
01.3_number_variant_coverage.R Variant counts by mutation type and region; per-exon sequence coverage across libraries.
01.4_overwiev_variant_effect.R Distribution of effect sizes (ΔLogitPSI bins) across mutation types and genomic regions; Fisher's exact and chi-squared tests.

02 — Heatmaps

Script Description
02.1_heatmap_585_exons.R ΔPSI heatmap across all exons, ordered by WT PSI (built in two phases: matrix construction then plotting).
02.2_all_heatmaps_and_wt_logo.R Per-exon heatmaps and WT sequence logos (Supplementary Figure 1).

03 — Mutational sensitivity

Script Description
03.1_mutational_sensitivity_WT_PSI.R Effect of WT PSI on mutational sensitivity, by mutation type and region.
03.2_mutational_sensitivity_exon_length.R Effect of exon length on mutational sensitivity; PSI loss below 30 nt.
03.3_mutational_sensitivity_splice_site.R Effect of splice site strength (MAxEntScan) on mutational sensitivity
03.4_mutational_sensitivity_region_mut_type.R Effect of position/WT nucleotide identity on mutational sensitivity

04 — Branch points (BP)

Script Description
04.1_branch_point_processing_data.R Process external BP datasets (Mercer 2015, Taggart 2017, LaBranchor).
04.2_branch_point_manual.R Mutational signature of manually annotated branch points.
04.3_branch_point_mercer_taggart_labranchor.R Mutational signature of preidcted branch points, external dataset comparisons, and Supplementary Table 7.

06 — Splicing cis-regulatory elements (SRE)

Script Description
06.1_cis_regulatory_element_mapping.R Map SRE positions (enhancer / silencer / overlap / neutral) from max/min ΔLogitPSI profiles.
06.2_preparing_clustering_files_regulatory_elements.R Build per-position SRE annotation matrix and ΔLogitPSI heatmap matrices for clustering.
06.3_clustering_regulatory_state.R Compute per-region SRE coverage, hierarchically cluster exons (Ward.D2, k = 6), assign cluster IDs.
06.4_state_description_overall.R SRE state composition across all exons and PSI groups (all / PSI = 20–80 / PSI < 20 / PSI > 80).
06.5_triangle_plot_regulatory_elements.R Ternary plots of per-exon SRE composition; correlation with WT PSI and exon length.
06.6_transition.R SRE state-transition rates vs. WT PSI and exon length, per region and transition type.
06.7_clustering_plot1.R Heatmaps of the SRE state matrix and ΔLogitPSI matrices ordered by cluster; per-cluster state-coverage bar charts.
06.8_clustering_plot2.R Cluster description plots, state-transition analysis per cluster, pairwise tests, summary tables.
06.9.1_SpliceMaps_main.R Splice maps for 30 curated example exons arranged in a 6-column grid.
06.9.2_SpliceMaps_all.R Splice maps for all exons (≥ 50 % coverage), two-panel per exon (Supplementary Figure 2).
06_shared.R Helper functions shared by 06.6 and 06.8 (calculate_state_transitions()). Not run directly.

07 — ClinVar benchmarking

Script Description
07_clinvar.R Bar charts and ROC curves benchmarking splicing effect calls against ClinVar classifications; run for all variants and for high-confidence annotations (status ≥ 2).

Outputs

Type Location
Figures (PNG) figures/<NN_group>/<NN.script_name>/
Result tables results/analysis/<NN_group>/<NN.script_name>/
Supplementary tables results/supplementary_tables/

Shared utilities

  • config.R — central path definitions (inputs, outputs, database files). Sourced by every script. Edit only if adding new database paths.
  • 06_shared.R — helper functions for the SRE clustering scripts; sourced explicitly by 06.6 and 06.8.