---
title: "DASC user guide (PDF)"
author: Haidong Yi†, Ayush T. Raman†, Han Zhang, Genevera Allen, Zhandong Liu
date: "`r BiocStyle::doc_date()`"
package: "`r BiocStyle::pkg_ver('DASC')`"
abstract: |
    Batch effects are one of the major source of technical variations in high 
    throughput studies such as omics profiling. It has been well established 
    that batch effects can be caused by different experimental platforms, 
    laboratory conditions, different sources of samples and personnel 
    differences. These differences can confound the outcomes of interest and 
    lead to spurious results. A critical input for batch correction algorithms 
    are the knowledge of batch factors, which in many cases are unknown or 
    inaccurate. Hence, the primary motivation of our paper is to detect hidden 
    batch factors that can be used in standard techniques to accurately capture 
    the relationship between expression and other modeled variables of 
    interest. Here, we present `r Biocpkg("DASC")`, a novel algorithm that is
    based on convex clustering and semi-NMF for the detection of unknown batch 
    effects.
output: 
    BiocStyle::pdf_document
vignette: |
    %\VignetteIndexEntry{DASC user guide} 
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding[utf8]{inputenc}
    %\VignetteKeywords{BatchEffects, RNASeq, GeneExpression, Normalization, 
        Preprocessing, QualityControl, StatisticalMethod}
---

```{r setup, echo=FALSE, results="hide", message=FALSE}
knitr::opts_chunk$set(tidy=FALSE, cache=TRUE, 
                        dev="png", message=FALSE, error=FALSE, warning=TRUE)
## Libraries
require("Biobase")
require("NMF")
require("cvxclustr")
```

# Getting started

`r Biocpkg("DASC")` is an R package distributed as part of the 
[Bioconductor](http://bioconductor.org) project. To install the package, start 
R and enter:

```{r installation, eval=FALSE}
source("http://bioconductor.org/biocLite.R")
biocLite("DASC")
```

# Introduction

`r Biocpkg("DASC")` is used for identifying batches and classifying samples 
into different batches in a high dimensional gene expression dataset. The 
batch information can be further used as a covariate in conjunction with other 
variables of interest among standard bioinformatics analysis like differential 
expression analysis.

## Citation info

If you use `r Biocpkg("DASC")` for your analysis, please cite it as here below. 
To cite package ‘DASC’ in publications use:

```
    @Manual{, 
        title = {DASC: Detecting hidden batch factors through data adaptive 
            adjustment for biological effects.},
        author = {Haidong Yi, Ayush T. Raman, Han Zhang, Genevera I. Allen and 
            Zhandong Liu},
        year = {2017},
        note = {R package version 0.1.0},
    }
```

# Quick Example

```{r, message=FALSE, results='hide', eval=TRUE}
library(DASC)
data("esGolub")
samples <- c(20,21,28,30)
dat <- exprs(esGolub)[1:100,samples]
pdat <- pData(esGolub)[samples,]

## use nrun = 50 or more for better convergence of results
res <- DASC(edata = dat, pdata = pdat, factor = pdat$Cell, method = 'ama',
                type = 3, lambda = 1, rank = 2:3, nrun = 5,
                annotation="esGolub Dataset")
#consensusmap(res)
#plot(res)
```

# Setting up the data

The first step in using `DASC` package is to properly format the data. For 
example, in case of gene expression data, it should be a matrix with features 
(genes, transcripts) in the rows and samples in the columns. `DASC` then 
requires the information for the variable of interest to model the gene 
expression data effectively.Variable of interest could be a genotype or 
treatment information.

## Stanford RNA-Seq Dataset

Below is an example of Stanford gene expression dataset (Chen et. al. PNAS, 
2015; Gilad et. al. F1000 Research, 2015). It is a filtered raw counts dataset 
which was published by Gilad et al. F1000 Research. 30% of genes with the 
lowest expression & mitochondrial genes were removed (Gilad et al.F1000 
Research).

```{r, message=FALSE, eval=TRUE}
## libraries
set.seed(99999)
library(DESeq2)
library(ggplot2)
library(pcaExplorer)

## dataset
rawCounts <- stanfordData$rawCounts
metadata <- stanfordData$metadata
```

```{r, message=FALSE, eval=TRUE}
## Using a smaller dataset
idx <- which(metadata$tissue %in% c("adipose", "adrenal", "sigmoid"))
rawCounts <- rawCounts[,idx]
metadata <- metadata[idx,]
```

```{r, message=FALSE, eval=TRUE}
head(rawCounts)
head(metadata)
```

# Batch detection using PCA Analysis

```{r, message=FALSE, eval=TRUE}
## Normalizing the dataset using DESeq2
dds <- DESeqDataSetFromMatrix(rawCounts, metadata, design = ~ species+tissue)
dds <- estimateSizeFactors(dds)
dat <- counts(dds, normalized = TRUE)
lognormalizedCounts <- log2(dat + 1)
```

```{r, message=FALSE, eval=TRUE}
## PCA plot using 
rld.dds <- rlog(dds)
pcaplot(rld.dds, intgroup=c("tissue","species"), ntop=1000, pcX=1, pcY=2)
```

In the PCA plot, PC1 shows the differences between the species. PC2 shows the 
differences between the species i.e. samples clustering based on tissues.

# Batch detection using DASC

```{r, message=FALSE, eval=TRUE}
res <- DASC(edata = dat, pdata = metadata, factor = metadata$tissue,
                method = 'ama', type = 3, lambda = 1, rank = 2:3, nrun = 10,
                annotation = 'Stanford Dataset')
```

```{r, message=FALSE, eval=TRUE}
## Consensus plot
consensusmap(res)
```

```{r, message=FALSE, eval=TRUE}
## Residual plot
plot(res)
```

```{r, message=FALSE, eval=TRUE}
## Batches -- dataset has 6 batches
sample.clust <- data.frame(sample.name = colnames(lognormalizedCounts), 
                            clust = as.vector(predict(res$fit$`2`)), 
                            batch = metadata$seqBatch)
ggplot(data = sample.clust, aes(x=c(1:6), y=clust, color=factor(clust))) + 
    geom_point(size = 4) + xlab("Sample Number") + ylab("Cluster Number")
```

Based on the above plots, we observe that the dataset has 2 batches. This can 
further be compared with the sequencing platform or `metadata$seqBatch`. The 
results suggest that differences in platform led to batch effects. Batch number 
can be used as another covariate, when differential expression analyses using 
`DESeq2`,`edgeR` or `limma` are performed.

# Session Info

```{r, message=FALSE}
sessionInfo()
```
