Cell type specific open chromatin signal matrix

Prerequisities

Some of UCSC tools https://genome.ucsc.edu/goldenPath/help/bigWig.html:

• bedGraphToBigWig

• bigWigMerge

• bigWigToBedGraph

• bedtools (http://quinlanlab.org/tutorials/bedtools/bedtools.html)\ R packages:

• tidyverse

• preprocessCore

• data.table

How to create open chromatin signal matrix:

1.-7. in /dataDownloadAndPreprocess folder

Set up path to the folder /dataDownloadAndPreprocess in your .bash_profile.

1. Download signal tracks from ENCODE

At ENCODE website apply criteria for file selection - hg19 / DNA accessibility / bigWig. Download files.txt.
The first line in files.txt contains the link to metadata connected to these files - download metadata.tsv.

In RStudio run: filterMetadata.R.
The output of filterMetadata.R is metadata_cells.tsv, and downloadCells.txt.

From terminal:

mkdir primaryCells
mv downloadCells.txt primaryCells
cd primaryCells
xargs -L 1 curl -O -L < downloadCells.txt
cd ..

2. Organize bigWig files into cell-specific subdirectories and merge

From RStudio run script: organizeFilesIntoCellFolders.R.
The script contains function moveBigWigs(cellMetadata, mainDir), which requires 2 inputs:

• name of the metadata file containing file name - cell type information (metadata_cells.tsv)
• name of a folder conatining downloaded bigWig files (/primary cells)

Within the directory provided to moveBigWigs a cell specific subdirectories are created and the bigWig files connected to the given cell type are moved into the subdirectory.
Followin step merges bigWig files coming from the same cell type. The output in form of bedGraph files is moved to a directory specified by used.

mkdir primaryCells_bedGraph
cd primaryCells
mergeBigWig.sh
cd ..

Following questions pop out:

What is the full path to the folder where outputs should be stored?
path_to_dataDownloadAndProcess/primaryCells_mergedBigWig

What is the full path to the file with chromosome sizes?
path_to_dataDownloadAndProcess/hg19chrom.sizes

If a folder contains a single bigWig file an error is generated In these folders run bigWigToBedGraph manually and move to a corresponding folder with all the other merged bedGraphs.

3. Download cell specific open chromatin BED files

At ENCODE website apply filtering criteia - DNA accessibility / hg19 / primary cell / bed narrowPeak.
Same as with the bigWig files, download the text document with links for BED file download (dataDownloadAndProcess/BED_files/files.txt) and diwnload the metadata from the first line (dataDownloadAndProcess/BED_files/metadata.tsv).
Run following script to select only trully hg19 BED files and have status released.

filterMetadata_BEDfiles.R

This script creates a new text file with filtered links: (dataDownloadAndProcess/BED_files/downloadBEDfiles.txt)
To get the BEd files run following from terminal:

cd BED_files
xargs -L 1 curl -O -L < downloadBEDfiles.txt

4. Create a set of all possible open chromatin regions across cell types

To create a universe of oll possible chromatin accessible regions run following command from the directory with all the downloaded BED files:

cat *.bed | sort -k1,1 -k2,2n | bedtools merge -i stdin > MasterPeaks.bed

You can now remove all the downloaded BEd files and keep only the MasterPeaks.bed. Create a 4th column in MasterPeaks.bed with names for individual peaks (e.g. chr_start_end) - otherwise and error will be generated in following step.

5. Assign cell specific signal values to the genomic regions defined in step 4

Place the MasterPeaks.bed file into a directory, where it will be the only BED file.
From the folder with final normalised bigWig files run following script:

cellSpecificity_bigWigOverBed.sh

Following question pops out. Give the full path to the folder containing MasterPeaks.bed - example bellow.

What is the folder with your BED files?
dataDownloadAndProcess/BED_files

A new directory will be created within a folder with bigWig files - /MasterPeaks_coverage. It contains coverage files for individual cell types in the predefined regions. The columns in the TAB files are following:

• name - name field from bed, which should be unique (the 4th column in BED file)
• size - size of bed (sum of exon sizes
• covered - # bases within exons covered by bigWig
• sum - sum of values over all bases covered
• mean0 - average over bases with non-covered bases counting as zeroes
• mean - average over just covered bases
• min - minimum observed in the area
• max - maximum observed in the area

6. Merge individual signal tracks into matrix

Run following script, where you must first set a path to the folder with coverage files (the TAB files generated by running cellSpecificity_bigWigOverBed.sh) - line 5 (variable folderName).

createOpenMatrix.R

The script creates two matrices - one with maximum coverage value over the given region and one with mean0 value over the given region. Rows are individual genomic regions, columns are individual cell types.

7. Normalize matrix

Final step of creating the cell specific open chromatin matrix is normalization. This is done by running normalizeOpenMatrix.R , where previously created matrix is passed to the variable rawMatrix. The script creates the open chromatin cell specific matrix in its final form, which is called meanCoverage_percentile99_01_quantNormalized_round4d.txt.
The normalization steps are following:

1. Set all values above 99th percentile for a given cell type to 1.
2. Normalize the rest of the values to range from 0 to 1.
3. Perform quantile normalization for cell types to be comparable.