04-13C-Exhaustion.Rmd

---
title: "C13-Exhaustion"
author: "Alexander Kirchmair"
params:
  results:    ../results/metabolomics
---


```{r setup, include=FALSE}
library(c13ms)
library(datamisc)
library(dplyr)
library(openxlsx)

dir.create("../data/metabolomics", showWarnings = FALSE)
dir.create(params$results, showWarnings = FALSE)
```


## Prepare data and metadata
```{r}
design <- read.xlsx("../data/metabolomics/13C_EXH_samplesheet.xlsx")
design$name <- with(design, paste(Batch, Sampletype, Celltype, Donor, Tracer, sep = "_"))
design$Donor <- factor(as.character(design$Donor), ordered = TRUE, levels = c("1", "2", "3"))
design$MioCells <- design$Cellnumber/10^6

intensities <- read.xlsx(("../data/metabolomics/13C_EXH_data.xlsx"), sheet = "intensities", rowNames = TRUE)
intensities <- intensities[,design$Sample]
rownames(design) <- design$name
colnames(intensities) <- design$name

intensities$metabolite <- rownames(intensities) |> strsplit(split = "_m") |> sapply(FUN = "[", 1)
intensities$label <- rownames(intensities) |> strsplit(split = "_m") |> sapply(FUN = "[", 2)
intensities$label <- paste0("m", intensities$label)

metabolites <- read.xlsx("../data/metabolomics/13C_EXH_data.xlsx", sheet = "metabolites")
rownames(metabolites) <- metabolites$ID

arrange(design, Celltype, Sampletype, Donor) |>
  ggdesign(columns = c(Celltype, Sampletype, Tracer, Donor, Batch), label = TRUE) |>
  saveplot(file = fp(params$results, "exp_design_exh"))

C13exh <- makeTracerExperiment(data = intensities[,c("metabolite", "label", design$name)],
                       colData = design,
                       metData = metabolites)

C13exh <- subset(C13exh, Sample != "MCF000187_GL01" & Batch != "A")# exclude based on QC

```


## Preprocessing

Preprocessing (data have already been filtered)
```{r}
C13exh@qcAssays$zero <- ifelse(C13exh@isoAssays$raw == 0, 0, Inf)
C13exh %<>% impute(split_by = ~ Celltype + Sampletype, assay = "raw", na = NULL, nan = 0)
C13exh %<>% correctIso(tracer = "C", assay =  "imp", tracer_purity = 0.99, mode = "negative", tol_error = 0.15, highres = TRUE)
```


Normalization
```{r }

## Cells
C13exhcells <- subset(C13exh, Sampletype == "cells")
C13exhcells %<>% normalize(method = ~ IS + MioCells + LEVEL + HKM, ISmet = "myr_d27_m0", assay = "corr")

## Medium
C13exhmedium <- subset(C13exh, Sampletype == "medium")
C13exhmedium %<>% normalize(method = ~ IS + LEVEL + HKM, ISmet = "myr_d27_m0", assay = "corr")

## Combine again
C13exh <- C13exhcells + C13exhmedium

```


Labelling
```{r}

C13exh_cells <- subset.TracerExperiment(C13exh, Sampletype == "cells")
C13exh_cells <- sumMets(C13exh_cells, assay = "norm", na_iso.rm = FALSE, qc_LOQ = "zero", thres_LOQ = 1, split_by = ~ Celltype, exclude = "myr_d27",
                        max_nafrac_per_group = 1,
                        min_rep_per_group = 2,
                        max_nafrac_per_met = 0.8,
                        min_groupfrac_per_iso = 0.5)

C13exh_medium <- subset.TracerExperiment(C13exh, Sampletype == "medium")
C13exh_medium <- sumMets(C13exh_medium, assay = "norm", na_iso.rm = FALSE, qc_LOQ = "zero", thres_LOQ = 1, split_by = ~ Celltype,
                        max_nafrac_per_group = 1,
                        min_rep_per_group = 2,
                        max_nafrac_per_met = 0.8,
                        min_groupfrac_per_iso = 0.5)

C13exh <- C13exh_cells + C13exh_medium
C13exh@metAssays$norm <- cjoin(C13exh_cells@metAssays$norm, C13exh_medium@metAssays$norm)[,rownames(C13exh@colData)]

C13exh <- MID(C13exh)
C13exh <- isoEnrichment(C13exh, na.rm = TRUE)
```


Clean data
```{r}
C13exh <- clean(C13exh, max_nafrac_per_group = 1, min_rep_per_group = 2, qc_LOQ = "zero", thres_LOQ = 1, split_by = ~ Sampletype, soft = TRUE)
C13exh %<>% clean(assay = "mid", new_assay = "mid_clean",
                  max_nafrac_per_group = 1, min_rep_per_group = 2, qc_LOQ = "zero", thres_LOQ = 1, split_by = ~ Sampletype, soft = TRUE)

```


# Differential abundance testing
```{r}

colData(C13exh)$Celltype <- factor(colData(C13exh)$Celltype)

contrasts <- list("TEXvsTEFF_cells" = list("Celltype" = c("TEX", "TEFF"), "Sampletype" = "cells", "Tracer" = "13C"),
                  "TEXvsTEFF_medium" = list("Celltype" = c("TEX", "TEFF"), "Sampletype" = "medium", "Tracer" = "13C"))

C13exh %<>% diffTest(contrasts = contrasts, formula = ~ Celltype, random = ~ 1 | Donor, type = "met", method = "lmm", assay = "norm")
C13exh %<>% diffTest(contrasts = contrasts, formula = ~ Celltype, random = ~ 1 | Donor, type = "iso", method = "lmm", assay = "norm")

C13exh %<>% diffTest(contrasts = contrasts, formula = ~ Celltype + (1|Donor), type = "met", method = "beta", assay = "frac")
C13exh %<>% diffTest(contrasts = contrasts, formula = ~ Celltype + (1|Donor), type = "iso", method = "beta", assay = "mid_clean")
```


```{r}
saveRDS(C13exh, "../data/C13exh.rds")
```