---
title: "03 - Unite CpGs and sample structure"
output: html_document
---

```{r setup, include=FALSE}
knitr::opts_chunk$set(eval = FALSE, message = FALSE, warning = FALSE)
library(methylKit); library(data.table); library(pheatmap)
```

# Unite: common CpGs across all 24 samples

```{r unite}
filt <- readRDS("methylRaw_filtered.rds")
meth <- unite(filt, destrand = FALSE)   # covered in ALL samples
saveRDS(meth, "meth_united.rds")
nrow(meth)   # expect 1,953,620
```

NOTE: a relaxed `unite(min.per.group = 4L)` can exceed a 48 GB vector limit on a
genome this size. The strict all-sample set is the primary object; per-contrast
subsets are re-united inside the differential-methylation notebooks.

# Correlation, clustering, PCA

```{r structure}
meta <- fread("sample_metadata.csv")
pm <- percMethylation(meth)                       # CpG x sample matrix
colnames(pm) <- meta$sampleID

# pairwise correlation
cc <- cor(pm, use = "pairwise.complete.obs")      # Pearson
saveRDS(cc, "sample_correlation.rds")

# PCA on the 200k most-variable CpGs
v <- matrixStats::rowVars(pm)
top <- order(v, decreasing = TRUE)[1:200000]
pca <- prcomp(t(pm[top, ]), center = TRUE, scale. = TRUE)
ve <- round(100 * pca$sdev^2 / sum(pca$sdev^2), 1)
pca_dt <- data.table(sampleID = meta$sampleID, meta[, .(ploidy, pH, group)],
                     PC1 = pca$x[,1], PC2 = pca$x[,2], PC3 = pca$x[,3], PC4 = pca$x[,4])
fwrite(pca_dt, "pca_coordinates.csv")
ve[1:4]   # variance explained; expect ~13.2, 7.8, 6.9, 5.0
```

Samples are NOT expected to cluster by ploidy or pH; PC1 explains only ~13% of
variance. This is the motivation for per-CpG testing: treatment effects are
localized, not genome-wide.

```{r heatmap}
ann <- data.frame(ploidy = meta$ploidy, pH = meta$pH, row.names = meta$sampleID)
ann_cols <- list(ploidy = c("2N"="#8CB369","3N"="#5B5F97"),
                 pH = c("high"="#F4A259","low"="#BC4B51"))
pheatmap(cc, annotation_col = ann, annotation_colors = ann_cols,
         clustering_method = "ward.D2", filename = "sample_correlation.png",
         width = 8, height = 7)
```