PTM Analysis - Sequence Logo • prophosqua

Introduction

This document performs sequence logo analysis on significantly regulated sites from DPA analysis. Sequence motifs help identify active kinases driving PTM changes.

desc <- switch(params$sheet,
  "DPA" = "**DPA (Differential PTM Abundance)**: Raw PTM signal changes. Sequence motifs may reflect both abundance and stoichiometry effects.",
  "DPU" = "**DPU (Differential PTM Usage)**: Protein-normalized changes. Sequence motifs reflect genuine kinase activity changes.",
  "CF" = "**CF (CorrectFirst)**: Alternative normalization. Sequence motifs reflect activity changes with different correction approach."
)
cat(desc)

DPA (Differential PTM Abundance): Raw PTM signal changes. Sequence motifs may reflect both abundance and stoichiometry effects.

Data Loading

if (pipeline_mode) {
  message("Loading data from: ", params$xlsx_file, " (sheet: ", params$sheet, ")")
  data <- readxl::read_xlsx(params$xlsx_file, sheet = params$sheet)
} else {
  # Vignette mode: use example data
  data("combined_test_diff_example", package = "prophosqua")
  data <- combined_test_diff_example
  message("Using example data from prophosqua package")
}

data_info <- tibble(
  Property = c("Mode", "Sheet", "Rows", "Contrasts"),
  Value = c(
    if (pipeline_mode) basename(params$xlsx_file) else "Example data",
    params$sheet,
    nrow(data), paste(unique(data$contrast), collapse = ", ")
  )
)
knitr::kable(data_info, caption = "Data Summary")

Data Summary
Property	Value
Mode	Example data
Sheet	DPA
Rows	105824
Contrasts	KO_vs_WT, KO_vs_WT_at_Early, KO_vs_WT_at_Late, KO_vs_WT_at_Uninfect

Filter Significant Sites

Filter sites with |log2FC| > 0.6 and FDR < 0.05.

# Use shared filtering function with sequence validation
significant_sites <- data |>
  dplyr::filter(!is.na(posInProtein)) |>
  filter_significant_sites(
    fdr_threshold = params$fdr,
    fc_threshold = params$fc,
    require_sequence = TRUE
  )

cat("Found", nrow(significant_sites), "significant sites\n")

## Found 7410 significant sites

Count Significant Sites

stopifnot("No significant sites found. Adjust fdr/log2fc thresholds in config." = nrow(significant_sites) > 0)

tx <- as.data.frame(with(significant_sites, table(contrast, regulation, modAA)))
capt <- paste0("Number of significantly regulated sites by contrast, residue, and regulation direction (", params$sheet, ").")

knitr::kable(tx, caption = capt)

Number of significantly regulated sites by contrast, residue, and regulation direction (DPA).
contrast	regulation	modAA	Freq
KO_vs_WT	downregulated	S	807
KO_vs_WT_at_Early	downregulated	S	785
KO_vs_WT_at_Late	downregulated	S	797
KO_vs_WT_at_Uninfect	downregulated	S	484
KO_vs_WT	upregulated	S	814
KO_vs_WT_at_Early	upregulated	S	812
KO_vs_WT_at_Late	upregulated	S	889
KO_vs_WT_at_Uninfect	upregulated	S	433
KO_vs_WT	downregulated	T	217
KO_vs_WT_at_Early	downregulated	T	177
KO_vs_WT_at_Late	downregulated	T	250
KO_vs_WT_at_Uninfect	downregulated	T	107
KO_vs_WT	upregulated	T	153
KO_vs_WT_at_Early	upregulated	T	170
KO_vs_WT_at_Late	upregulated	T	177
KO_vs_WT_at_Uninfect	upregulated	T	58
KO_vs_WT	downregulated	Y	12
KO_vs_WT_at_Early	downregulated	Y	17
KO_vs_WT_at_Late	downregulated	Y	13
KO_vs_WT_at_Uninfect	downregulated	Y	9
KO_vs_WT	upregulated	Y	55
KO_vs_WT_at_Early	upregulated	Y	78
KO_vs_WT_at_Late	upregulated	Y	76
KO_vs_WT_at_Uninfect	upregulated	Y	20

Validate Sequence Window

Ensure the central residue matches the modified amino acid.

# Use shared validation function
significant_sites <- validate_sequence_window(significant_sites)

cat("After validation:", nrow(significant_sites), "sites remain\n")

## After validation: 7261 sites remain

Sequence Logo Analysis

Generate Sequence Logos

seq_list <- significant_sites |>
  dplyr::mutate(.grp = paste(contrast, regulation, sep = "_")) |>
  dplyr::group_by(.grp) |>
  dplyr::summarize(
    seqs = list(toupper(SequenceWindow)),
    .groups = "drop"
  ) |>
  with(setNames(seqs, .grp))

if (length(seq_list) > 0) {
  ggseqlogo(
    seq_list,
    ncol = 2,
    seq_type = "aa",
    method = "probability"
  )
} else {
  cat("No significant sites found for sequence logo generation.\n")
}

Sequence logos for significantly regulated sites by contrast and regulation direction.

Difference Logo Analysis

Visualize the difference in amino acid enrichment between upregulated and downregulated sites.

p_diff <- prophosqua::plot_diff_logo(significant_sites)

if (!is.null(p_diff)) {
  print(p_diff)
} else {
  cat("No contrasts with both upregulated and downregulated sites found for difference logo.\n")
}

Difference Logos (Upregulated - Downregulated)

Results Summary

summary_info <- tibble(
  Metric = c("Analysis Type", "FDR Threshold", "FC Threshold",
             "Total Significant Sites", "Upregulated", "Downregulated"),
  Value = c(params$sheet, params$fdr, params$fc,
            nrow(significant_sites),
            sum(significant_sites$regulation == "upregulated"),
            sum(significant_sites$regulation == "downregulated"))
)
knitr::kable(summary_info, caption = "Analysis Summary")

Analysis Summary
Metric	Value
Analysis Type	DPA
FDR Threshold	0.05
FC Threshold	0.6
Total Significant Sites	7261
Upregulated	3666
Downregulated	3595

Session Info

sessionInfo()

## R version 4.5.2 (2025-10-31)
## Platform: aarch64-apple-darwin20
## Running under: macOS Tahoe 26.3
## 
## Matrix products: default
## BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
## LAPACK: /Library/Frameworks/R.framework/Versions/4.5-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.1
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## time zone: Europe/Zurich
## tzcode source: internal
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] prophosqua_0.3.0 readxl_1.4.5     ggseqlogo_0.2.2  dplyr_1.2.0     
## 
## loaded via a namespace (and not attached):
##  [1] gtable_0.3.6       jsonlite_2.0.0     compiler_4.5.2     tidyselect_1.2.1  
##  [5] jquerylib_0.1.4    systemfonts_1.3.1  scales_1.4.0       textshaping_1.0.4 
##  [9] yaml_2.3.12        fastmap_1.2.0      ggplot2_4.0.2      R6_2.6.1          
## [13] labeling_0.4.3     patchwork_1.3.2    generics_0.1.4     knitr_1.51        
## [17] forcats_1.0.1      htmlwidgets_1.6.4  tibble_3.3.1       bookdown_0.46     
## [21] desc_1.4.3         bslib_0.9.0        pillar_1.11.1      RColorBrewer_1.1-3
## [25] rlang_1.1.7        cachem_1.1.0       xfun_0.55          S7_0.2.1          
## [29] fs_1.6.6           sass_0.4.10        otel_0.2.0         cli_3.6.5         
## [33] withr_3.0.2        pkgdown_2.2.0      magrittr_2.0.4     digest_0.6.39     
## [37] grid_4.5.2         lifecycle_1.0.5    vctrs_0.7.1        evaluate_1.0.5    
## [41] glue_1.8.0         cellranger_1.1.0   farver_2.1.2       ragg_1.5.0        
## [45] purrr_1.2.1        rmarkdown_2.30     tools_4.5.2        pkgconfig_2.0.3   
## [49] htmltools_0.5.9

PTM Analysis - Sequence Logo

DPA Analysis

FGCZ

17 February, 2026