16-remove-conf-reads.Rmd

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output: html_document
---

## Dealing with confounders (Reads)

```{r, echo=FALSE}
library(knitr)
opts_chunk$set(fig.align = 'center')
```

```{r, message=FALSE, warning=FALSE}
library(scRNA.seq.funcs)
library(RUVSeq)
library(scater)
library(SingleCellExperiment)
library(scran)
library(kBET)
library(sva) # Combat
library(edgeR)
set.seed(1234567)
options(stringsAsFactors = FALSE)
reads <- readRDS("tung/reads.rds")
reads.qc <- reads[rowData(reads)$use, colData(reads)$use]
endog_genes <- !rowData(reads.qc)$is_feature_control
erccs <- rowData(reads.qc)$is_feature_control

qclust <- quickCluster(reads.qc, min.size = 30)
reads.qc <- computeSumFactors(reads.qc, sizes = 15, clusters = qclust)
reads.qc <- normalize(reads.qc)
```

### Remove Unwanted Variation

#### RUVg

```{r, message=FALSE, warning=FALSE}
ruvg <- RUVg(counts(reads.qc), erccs, k = 1)
assay(reads.qc, "ruvg1") <- log2(
    t(t(ruvg$normalizedCounts) / colSums(ruvg$normalizedCounts) * 1e6) + 1
)
ruvg <- RUVg(counts(reads.qc), erccs, k = 10)
assay(reads.qc, "ruvg10") <- log2(
    t(t(ruvg$normalizedCounts) / colSums(ruvg$normalizedCounts) * 1e6) + 1
)
```

#### RUVs

```{r}
scIdx <- matrix(-1, ncol = max(table(reads.qc$individual)), nrow = 3)
tmp <- which(reads.qc$individual == "NA19098")
scIdx[1, 1:length(tmp)] <- tmp
tmp <- which(reads.qc$individual == "NA19101")
scIdx[2, 1:length(tmp)] <- tmp
tmp <- which(reads.qc$individual == "NA19239")
scIdx[3, 1:length(tmp)] <- tmp
cIdx <- rownames(reads.qc)
ruvs <- RUVs(counts(reads.qc), cIdx, k = 1, scIdx = scIdx, isLog = FALSE)
assay(reads.qc, "ruvs1") <- log2(
    t(t(ruvs$normalizedCounts) / colSums(ruvs$normalizedCounts) * 1e6) + 1
)
ruvs <- RUVs(counts(reads.qc), cIdx, k = 10, scIdx = scIdx, isLog = FALSE)
assay(reads.qc, "ruvs10") <- log2(
    t(t(ruvs$normalizedCounts) / colSums(ruvs$normalizedCounts) * 1e6) + 1
)
```

#### Combat

```{r, eval = TRUE, message = FALSE}
combat_data <- logcounts(reads.qc)
mod_data <- as.data.frame(t(combat_data))
# Basic batch removal
mod0 = model.matrix(~ 1, data = mod_data) 
# Preserve biological variability
mod1 = model.matrix(~ reads.qc$individual, data = mod_data) 
# adjust for total genes detected
mod2 = model.matrix(~ reads.qc$total_features, data = mod_data)
assay(reads.qc, "combat") <- ComBat(
    dat = t(mod_data), 
    batch = factor(reads.qc$batch), 
    mod = mod0,
    par.prior = TRUE,
    prior.plots = FALSE
)
```

__Exercise 1__

```{r, eval = TRUE, echo = FALSE, message = FALSE}
assay(reads.qc, "combat_tf") <- ComBat(
    dat = t(mod_data), 
    batch = factor(reads.qc$batch), 
    mod = mod2,
    par.prior = TRUE,
    prior.plots = FALSE
)
```

#### mnnCorrect 

```{r}
do_mnn <- function(data.qc) {
    batch1 <- data.qc[, data.qc$replicate == "r1"]
    batch2 <- data.qc[, data.qc$replicate == "r2"]
    batch3 <- data.qc[, data.qc$replicate == "r3"]
    
    if (ncol(batch2) > 0) {
        x = mnnCorrect(
          logcounts(batch1), logcounts(batch2), logcounts(batch3),  
          k = 20,
          sigma = 0.1,
          cos.norm = TRUE,
          svd.dim = 2
        )
        return(cbind(x$corrected[[1]], x$corrected[[2]], x$corrected[[3]]))
    } else {
        x = mnnCorrect(
          logcounts(batch1), logcounts(batch3),  
          k = 20,
          sigma = 0.1,
          cos.norm = TRUE,
          svd.dim = 2
        )
        return(cbind(x$corrected[[1]], x$corrected[[2]]))
    }
}

indi1 <- do_mnn(reads.qc[, reads.qc$individual == "NA19098"])
indi2 <- do_mnn(reads.qc[, reads.qc$individual == "NA19101"])
indi3 <- do_mnn(reads.qc[, reads.qc$individual == "NA19239"])

assay(reads.qc, "mnn") <- cbind(indi1, indi2, indi3);

# For a balanced design: 
#assay(reads.qc, "mnn") <- mnnCorrect(
#    list(B1 = logcounts(batch1), B2 = logcounts(batch2), B3 = logcounts(batch3)),  
#    k = 20,
#    sigma = 0.1,
#    cos.norm = TRUE,
#    svd.dim = 2
#)
```

#### GLM

```{r}
glm_fun <- function(g, batch, indi) {
  model <- glm(g ~ batch + indi)
  model$coef[1] <- 0 # replace intercept with 0 to preserve reference batch.
  return(model$coef)
}
effects <- apply(
    logcounts(reads.qc), 
    1, 
    glm_fun, 
    batch = reads.qc$batch, 
    indi = reads.qc$individual
)
corrected <- logcounts(reads.qc) - t(effects[as.numeric(factor(reads.qc$batch)), ])
assay(reads.qc, "glm") <- corrected
```

__Exercise 2__

```{r, echo=FALSE}
glm_fun1 <- function(g, batch) {
  model <- glm(g ~ batch)
  model$coef[1] <- 0 # replace intercept with 0 to preserve reference batch.
  return(model$coef)
}

do_glm <- function(data.qc) {
    effects <- apply(
        logcounts(data.qc), 
        1, 
        glm_fun1, 
        batch = data.qc$batch
    )
    corrected <- logcounts(data.qc) - t(effects[as.numeric(factor(data.qc$batch)), ])
    return(corrected)
}
indi1 <- do_glm(reads.qc[, reads.qc$individual == "NA19098"])
indi2 <- do_glm(reads.qc[, reads.qc$individual == "NA19101"])
indi3 <- do_glm(reads.qc[, reads.qc$individual == "NA19239"])

assay(reads.qc, "glm_indi") <- cbind(indi1, indi2, indi3);
```

#### Effectiveness 1

```{r}
for(n in assayNames(reads.qc)) {
    print(
        plotPCA(
            reads.qc[endog_genes, ],
            colour_by = "batch",
            size_by = "total_features",
            shape_by = "individual",
            exprs_values = n
        ) +
        ggtitle(n)
    )
}
```

#### Effectiveness 2

```{r}
res <- list()
for(n in assayNames(reads.qc)) {
	res[[n]] <- suppressWarnings(calc_cell_RLE(assay(reads.qc, n), erccs))
}
par(mar=c(6,4,1,1))
boxplot(res, las=2)
```

#### Effectiveness 3

```{r}
for(n in assayNames(reads.qc)) {
    print(
        plotQC(
            reads.qc[endog_genes, ],
            type = "expl",
            exprs_values = n,
            variables = c(
                "total_features",
                "total_counts",
                "batch",
                "individual",
                "pct_counts_ERCC",
                "pct_counts_MT"
            )
        ) +
        ggtitle(n)
    )
}
```

#### Effectiveness 4

```{r, message = FALSE}
compare_kBET_results <- function(sce){
    indiv <- unique(sce$individual)
    norms <- assayNames(sce) # Get all normalizations
    results <- list()
    for (i in indiv){ 
        for (j in norms){
            tmp <- kBET(
                df = t(assay(sce[,sce$individual== i], j)), 
                batch = sce$batch[sce$individual==i], 
                heuristic = TRUE, 
                verbose = FALSE, 
                addTest = FALSE, 
                plot = FALSE)
            results[[i]][[j]] <- tmp$summary$kBET.observed[1]
        }
    }
    return(as.data.frame(results))
}

eff_debatching <- compare_kBET_results(reads.qc)
```

```{r, message = FALSE}
require("reshape2")
require("RColorBrewer")
# Plot results
dod <- melt(as.matrix(eff_debatching),  value.name = "kBET")
colnames(dod)[1:2] <- c("Normalisation", "Individual")

colorset <- c('gray', brewer.pal(n = 9, "RdYlBu"))

ggplot(dod, aes(Normalisation, Individual, fill=kBET)) +  
    geom_tile() +
    scale_fill_gradient2(
        na.value = "gray",
        low = colorset[2],
        mid=colorset[6],
        high = colorset[10],
        midpoint = 0.5, limit = c(0,1)) +
    scale_x_discrete(expand = c(0, 0)) +
    scale_y_discrete(expand = c(0, 0)) + 
    theme(
        axis.text.x = element_text(
            angle = 45, 
            vjust = 1, 
            size = 12, 
            hjust = 1
        )
    ) + 
    ggtitle("Effect of batch regression methods per individual")
```

#### sessionInfo()

```{r echo=FALSE}
sessionInfo()
```