General Information

This code was created by Francisco Avila Cobos.

There is a webtool implementation available at http://zipperplot.cmgg.be

Feel free to provide feedback/inform about issues by sending an email to: Francisco.AvilaCobos (at) UGent (dot) be

Citing the Zipper plot

If used, please cite our work:

Avila Cobos, F. et al. Zipper plot: visualizing transcriptional activity of genomic regions. BMC Bioinformatics 18, 231 (2017).

Step 1: Installing software requirements

BEDTools v2.26.0:

Option 1)

• First: go to your browser and download:

https://github.com/arq5x/bedtools2/archive/6f9c61fa34c077082ca9f8785992f3804c210e5d.zip

• Second: don't forget to move the .zip file you have just downloaded to the folder where you want to run the analysis (e.g. "your_folder").

• Third: Type on the command line:

$ cd your_folder
$ unzip 6f9c61fa34c077082ca9f8785992f3804c210e5d.zip
$ cd bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d
$ make
$ cd ..

NOTE: Once this is complete, the path to bedtools should be:

/your_folder/bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d/bin/bedtools

Where "/your_folder/" could be, for example, "/Users/favilaco/Desktop/"

Option 2)

• Type on the command line:

$ cd your_folder
$ wget https://github.com/arq5x/bedtools2/archive/6f9c61fa34c077082ca9f8785992f3804c210e5d.zip
$ unzip 6f9c61fa34c077082ca9f8785992f3804c210e5d.zip
$ cd bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d
$ make
$ cd ..

NOTE: Once this is complete, the path to bedtools should be:

/your_folder/bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d/bin/bedtools

Where "/your_folder/" could be, for example, "/Users/favilaco/Desktop/"

R packages:

NOTE: R version 3.3.1 should be already installed

Required packages: ggplot2_2.2.1, R.utils_2.5.0, data.table_1.10.4, gridExtra_2.2.1

• Paste and run the following commands in R:

> packages <- c("ggplot2","R.utils","data.table","gridExtra")
> for (i in packages){ install.packages(i, character.only = TRUE)}

Step 2: Cloning the Zipper plot Github repository to your computer/server

Go to the folder in which you want to run your analysis and type on the command line:

$ cd your_folder
$ git clone https://github.com/favilaco/Zipper_plot

Step 3: Download the filtered and pre-processed input files:

Move to the folder Zipper_plot and, once you are there, download the following file by running the following on the command line:

$ cd your_folder/Zipper_plot
$ wget https://www.dropbox.com/s/t6g3fb8atcwjp8c/InputFiles_ZP.zip

Unzip "InputFiles_ZP.zip" from the command line, by running the commands:

$ unzip InputFiles_ZP.zip

Step 4: Preparing the input file with the correct format:

Please create a text file containing 4 tab separated columns as in this box (1 line per genomic feature):

chr1	17383802	-	lnc-name1
chr2	1936171	+	lnc-name2
...

NOTE: Please do not forget to include "chr" as part of your chromosome name.

As an example, you can download an example file (input.txt) by running the following on the command line:

$ cd your_folder/Zipper_plot
$ wget https://www.dropbox.com/s/cjvh9b414qpoadg/input.txt

EXAMPLE USAGE:

Once you completed all the above steps and have in the same folder: the unzipped version of "InputFiles_ZP", your input file and the R scripts, you can run one of the following four options [see our manuscript for detailed information]:

FANTOM5 (CAGE-seq) with ALL sample types:

$ cd your_folder/Zipper_plot
$ Rscript ZP_CAGE_ALL.R input.txt /usr/bin/bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d/bin/bedtools 5000 100 0 output_1_CAGE_ALL

Where the parameters after "Rscript" (in order): Script_file, input_file; path_to_bedtools, Zipper_width_in_nucleotides, Number_permutations_for_AUZpval, tpm_threshold and output_name

FANTOM5 (CAGE-seq) with 1 sample type :

$ cd your_folder/Zipper_plot
$ Rscript ZP_CAGE_1tissue.R input.txt /usr/bin/bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d/bin/bedtools 5000 100 0 CNhs10723.10400-106A4 OFF output_2_CAGE_CNhs10723.10400-106A4

Where the parameters after "Rscript" (in order): Script_file, input_file; path_to_bedtools, Zipper_width_in_nucleotides, Number_permutations_for_AUZpval, tpm_threshold, sample_type, TSS_pval (ON/OFF) and output_name

NOTE: ALL possible sample types can be found in the file "ManualCuration_2column.txt"

• For example: "CNhs10625.10019-101D1", which represents "(lung, adult, pool1)"

Roadmap (ChIP-seq/DNase-seq) with ALL sample types:

$ cd your_folder/Zipper_plot
$ Rscript ZP_ROADMAP_ALL.R input.txt /usr/bin/bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d/bin/bedtools 5000 100 H3K4me3 narrowPeak output_3_H3K4me3_narrow_ALL"

Where the parameters after "Rscript" (in order): Script_file, input_file; path_to_bedtools, Zipper_width_in_nucleotides, Number_permutations_for_AUZpval, mark, peak_type and output_name

You can select:

1. narrowPeak, broadPeak or gappedPeak

2. H3K4me1, H3K4me2, H3K4me3, H3K36me3, H3K79me2, H4K20me1, H3K9ac, H3K14ac, H3K27ac or DNaseI

Roadmap (ChIP-seq/DNase-seq) with 1 sample type:

$ cd your_folder/Zipper_plot
$ Rscript ZP_ROADMAP_1tissue.R input.txt /usr/bin/bedtools2-6f9c61fa34c077082ca9f8785992f3804c210e5d/bin/bedtools 5000 100 E096-H3K4me3 narrowPeak OFF output_4_H3K4me3_narrow_E096

Where the parameters after "Rscript" (in order): Script_file, input_file; path_to_bedtools, Zipper_width_in_nucleotides, Number_permutations_for_AUZpval, tissue-mark, peak_type, TSS_pval (ON/OFF) and output_name

You can select:

1. narrowPeak, broadPeak or gappedPeak

2. H3K4me1, H3K4me2, H3K4me3, H3K36me3, H3K79me2, H4K20me1, H3K9ac, H3K14ac, H3K27ac or DNaseI

3. Any tissue that can be found in the file "ROADMAP_info.txt".

• For example: "E096" (lung)

Update:

Depending on the R and data.table versions you are using, you may get the following warning message (that you can safely ignore) while the output is being generated:

"Invalid .internal.selfref detected and fixed by taking a (shallow) copy of the data.table so that := can add this new column by reference. [...] Also, in R<=v3.0.2, list(DT1,DT2) copied the entire DT1 and DT2 (R's list() used to copy named objects); please upgrade to R>v3.0.2 if that is biting."