---
title: "Updating methylSig code"
author: "Raymond G. Cavalcante"
date: "`r Sys.Date()`"
output: BiocStyle::html_document
vignette: >
  %\VignetteIndexEntry{Updating methylSig code}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
---

```{r, include = FALSE}
knitr::opts_chunk$set(
    collapse = TRUE,
    comment = "#>"
)
```

```{r setup}
library(methylSig)
```

# Introduction

The purpose of this vignette is to show users how to retrofit their `methylSig` < 0.99.0 code to work with the refactor in version 0.99.0 and later.

# Reading Data

## Old methylSig

In versions < 0.99.0 of `methylSig`, the `methylSigReadData()` function read Bismark coverage files, Bismark genome-wide CpG reports, or MethylDackel bedGraphs. Additionally, users could destrand the data, filter by coverage, and filter SNPs.

```{r eval = FALSE}
meth = methylSigReadData(
    fileList = files,
    pData = pData,
    assembly = 'hg19',
    destranded = TRUE,
    maxCount = 500,
    minCount = 10,
    filterSNPs = TRUE,
    num.cores = 1,
    fileType = 'cytosineReport')
```

## New methylSig

In versions >= 0.99.0 of `methylSig`, the user should read data with `bsseq::read.bismark()` and then apply functions that were once bundled within `methylSigReadData()`.

```{r read}
files = c(
    system.file('extdata', 'bis_cov1.cov', package='methylSig'),
    system.file('extdata', 'bis_cov2.cov', package='methylSig')
)

bsseq_stranded = bsseq::read.bismark(
    files = files,
    colData = data.frame(row.names = c('test1','test2')),
    rmZeroCov = FALSE,
    strandCollapse = FALSE
)
```

After reading data, filter by coverage. Note, we are changing our dataset to something we can use with the downstream functions.

```{r filter_by_coverage}
# Load data for use in the rest of the vignette
data(BS.cancer.ex, package = 'bsseqData')
bs = BS.cancer.ex[1:10000]

bs = filter_loci_by_coverage(bs, min_count = 5, max_count = 500)
```

If the locations of C-to-T and G-to-A SNPs are known, or some other set of location should be removed:

```{r filter_by_location}
# Construct GRanges object
remove_gr = GenomicRanges::GRanges(
    seqnames = c('chr21', 'chr21', 'chr21'),
    ranges = IRanges::IRanges(
        start = c(9411552, 9411784, 9412099),
        end = c(9411552, 9411784, 9412099)
    )
)

bs = filter_loci_by_location(bs = bs, gr = remove_gr)
```

# Tiling Data

## Old methylSig

In versions < 0.99.0 of `methylSig`, the `methylSigTile()` function combined aggregating CpG data over pre-defined tiles and genomic windows.

```{r eval = FALSE}
# For genomic windows, tiles = NULL
windowed_meth = methylSigTile(meth, tiles = NULL, win.size = 10000)

# For pre-defined tiles, tiles should be a GRanges object.
```

## New methylSig

In versions >= 0.99.0 of `methylSig`, tiling is separated into two functions, `tile_by_regions()` and `tile_by_windows()`. Users should chooose one or the other.

```{r tile_by_windows}
windowed_bs = tile_by_windows(bs = bs, win_size = 10000)
```

```{r tile_by_regions}
# Collapsed promoters on chr21 and chr22
data(promoters_gr, package = 'methylSig')

promoters_bs = tile_by_regions(bs = bs, gr = promoters_gr)
```

# Testing

## MethylSig Test

### Old methylSig

In versions < 0.99.0 of `methylSig`, the `methylSigCalc` function had a `min.per.group` parameter to determine how many samples per group had to have coverage in order to be tested.

```{r eval = FALSE}
result = methylSigCalc(
    meth = meth,
    comparison = 'DR_vs_DS',
    dispersion = 'both',
    local.info = FALSE,
    local.winsize = 200,
    min.per.group = c(3,3),
    weightFunc = methylSig_weightFunc,
    T.approx = TRUE,
    num.cores = 1)
```

### New methylSig

In versions >= 0.99.0 of `methylSig`, the `min.per.group` functionality is performed by a separate function `filter_loci_by_group_coverage()`. Also note the change in form to define dispersion calculations, and the use of local information.

```{r filter_by_group_coverage}
# Look a the phenotype data for bs
bsseq::pData(bs)

# Require at least two samples from cancer and two samples from normal
bs = filter_loci_by_group_coverage(
    bs = bs,
    group_column = 'Type',
    c('cancer' = 2, 'normal' = 2))
```

After removing loci with insufficient information, we can now use the `diff_methylsig()` test.

```{r diff_methylsig}
# Test cancer versus normal with dispersion from both groups
diff_gr = diff_methylsig(
    bs = bs,
    group_column = 'Type',
    comparison_groups = c('case' = 'cancer', 'control' = 'normal'),
    disp_groups = c('case' = TRUE, 'control' = TRUE),
    local_window_size = 0,
    t_approx = TRUE,
    n_cores = 1)
```

## DSS Test

### Old methylSig

In versions < 0.99.0 of `methylSig`, the `methylSigDSS` function also had a `min.per.group` parameter to determine how many samples per group had to have coverage. Users also couldn't specify which methylation groups to recover. The form of `design`, `formula`, and `contrast`, remain the same in versions >= 0.99.0.

```{r eval = FALSE}
contrast = matrix(c(0,1), ncol = 1)
result_dss = methylSigDSS(
    meth = meth,
    design = design1,
    formula = '~ group',
    contrast = contrast,
    group.term = 'group',
    min.per.group=c(3,3))
```

### New methylSig

In versions >= 0.99.0 of `methylSig`, the single `methylSigDSS()` function is replaced by a fit function `diff_dss_fit()` and a test functiotn `diff_dss_test()`. As with `diff_methylsig()`, users should ensure enough samples have sufficient coverage with the `filter_loci_by_group_coverage()` function. The `design` and `formula` are unchanged in their forms.

If a continuous covariate is to be tested, `filter_loci_by_group_coverage()` should be skipped, as there are no groups. In prior versions of `methylSigDSS()`, this was not possible, and the group constraints were incorrectly applied prior to testing on a continuous covariate.

```{r filter_by_group_coverage2, eval = FALSE}
# IF NOT DONE PREVIOUSLY
# Require at least two samples from cancer and two samples from normal
bs = filter_loci_by_group_coverage(
    bs = bs,
    group_column = 'Type',
    c('cancer' = 2, 'normal' = 2))
```

```{r diff_dss_fit_simple}
# Test the simplest model with an intercept and Type
diff_fit_simple = diff_dss_fit(
    bs = bs,
    design = bsseq::pData(bs),
    formula = as.formula('~ Type'))
```

The `contrast` parameter is also changed in its form. Note the, additional parameters to specify how to recover group methylation. `methylation_group_column` and `methylation_groups` should be specified for group versus group comparisons. For continuous covariates, `methylation_group_column` is sufficient, and the samples will be grouped into top/bottom 25 percentile based on the continuous covariate column name given in `methylation_group_column`.

```{r diff_dss_test_simple}
# Test the simplest model for cancer vs normal
# Note, 2 rows corresponds to 2 columns in diff_fit_simple$X
simple_contrast = matrix(c(0,1), ncol = 1)

diff_simple_gr = diff_dss_test(
    bs = bs,
    diff_fit = diff_fit_simple,
    contrast = simple_contrast,
    methylation_group_column = 'Type',
    methylation_groups = c('case' = 'cancer', 'control' = 'normal'))
```

# Session Info

```{r sessionInfo}
sessionInfo()
```
