Counter RnAseq Window

Counter RNA seq Window (CRAW) compute and visualize the coverage of RNA seq experiment.

There are 3 ways to use craw:

• by install the standalone python scripts
• by using docker image
• by using singularity image

Installation

Requirements

• python >= 3.5
• psutil >= 5.6
• pysam == 0.15.2
• pandas >= 0.24
• scipy >= 0.16.1
• numpy >= 1.16
• matplotlib >= 3.0
• pillow >= 5.4

From package

using pip

pip install craw

if you use virtualenv do not forget to configure the matplotlib backend

Notes for MacOS

On MacOS install python > 3 from image on http://python.org . Then install craw using pip

pip3 install craw

craw will be installed in /Library/Framework/Python.Framework/Version/3.6/ So if you want to use directly craw_coverage and craw_htmp just create a symbolic linc like this

ln -s /Library/Framework/Python.Framework/Version/3.6/bin/craw_coverage /usr/local/bin/craw_coverage
ln -s /Library/Framework/Python.Framework/Version/3.6/bin/craw_htmp /usr/local/bin/craw_htmp

The craw documentation (html and pdf) is located in /Library/Framework/Python.Framework/Version/3.6/share/craw/

From repository

clone the project and install with the setup.py

git clone https://gitlab.pasteur.fr/bneron/craw.git

cd craw

python3 setup.py sdist
pip3 install dist/craw-master-devxxxxx.tar.gz

Testing my installation

The release come from with some unit and functional tests. to test if everything work fine.

cd craw
python3 tests/run_tests.py -vv

This step is only available from the sources (a clone of the repository or a tarball release). You cannot perform tests if you installed craw from pypi (pip install craw)

Using Docker Image

Docker images are available. The two scripts are accessible through the sub-command coverage or htmp. For instance to use the latest version of craw_htmp

docker pull c3bi/craw
docker run -v$PWD:/root -it c3bi/craw coverage --bam foo.bam --annot foo.annot --ref-col 'Position' --before 3 --after 5 --out foo.cov
docker run -v$PWD:/root -it c3bi/craw htmp --size raw --out foo.png  foo.cov

note:

In docker the interactive htmp output is not available.
So you must specify the --out option

Using Singularity Image

Singularity images are available. The two scripts are accessible through the sub-command coverage or htmp. For instance to use the latest version of craw_htmp

Pull the image locally

singularity pull --name craw shub://C3BI-pasteur-fr/craw

Then run it

./craw coverage --bam foo.bam --annot foo.annot --ref-col 'Position' --before 3 --after 5 --out foo.cov
./craw htmp --size raw --out foo.png  foo.cov

or

singularity run craw htmp --size raw --out foo.png  foo.cov

note:
Instead of Docker images, in Singularity images the interactive output is available.

Quickstart

A detailed documentation is available

• online:
• installed along craw in INSTALL_DIR/share/craw/doc/(html|pdf)

Inputs / Outputs

craw_coverage

Inputs

bam file

craw_coverage need a file of alignment reads called bam file. a bam file is a short DNA sequence read alignments in the Binary Alignment/Map format (.bam). craw_coverage needs also the corresponding index file (bai). The index file must be located beside the bam file with the same name instead to have the .bam extension it end by .bai extension. If you have not the index file you have to create it.

To index a bam file you need samtools. The command line is

samtools index file.bam

For more explanation see http://www.htslib.org/doc/ .

wig file

craw_coverage can compute coverage also from wig file see https://wiki.nci.nih.gov/display/tcga/wiggle+format+specification and http://genome.ucsc.edu/goldenPath/help/wiggle.html . for format specifications. Compare d to these specifications craw support coverages on both strands. the positive coverages scores are on the forward strand whereas the negative ones are on the reverse strand.

track type=wiggle_0 name="demo" color=96,144,246 altColor=96,144,246 autoScale=on  graphType=bar
variableStep chrom=chrI span=1
72      12.0000
73      35.0000
74      70.0000
75      127.0000
...
72      -88.0000
73      -42.0000
74      -12.0000
75      -1.0000

In the example above the coverage on the Chromosome I for the positions 72, 73, 74, 75 are 12, 35, 70, 127 on the forward strand and 88, 42, 12, 1 on the reverse strand.

The --bam and --wig options are mutually exclusive but one of these option is required.

annotation file

The annotation file is a tsv file. It's mean that it is a text file with value separated by tabulation (not spaces). The first line of the file must be the name of the columns the other lines the values. Each line represent a row.

name    gene    chromosome      strand  Position
YEL072W RMD6    chrV    +       14415
YEL071W DLD3    chrV    +       17845
YEL070W DSF1    chrV    +       21097

All lines starting with '#' character will be ignored.

# This is the annotation file for Wild type
# bla bla ...
name    gene    chromosome      strand  Position
YEL072W RMD6    chrV    +       14415
YEL071W DLD3    chrV    +       17845
YEL070W DSF1    chrV    +       21097

mandatory columns

There is 3 mandatory columns in the annotation file.

columns with fixed name

two with a fixed name:

• strand indicate on which strand is located the region of interest. The authorized values for this columns are +/- , 1/-1 or for/rev.
• chromosome the chromosome name where is located the region of interest.

columns with variable name

In addition of these two columns the column to define the position of reference is mandatory too, but the name of this column can be specified by the user. If it's not craw_coverage will use a column name 'position'.

If we want to compute coverage on variable window size, 2 extra columns whose name must be specified by the user by the following option:

• --start-col to define the beginning of the window (this position is included in the window)

• --stop-col to define the end of the window (this position is included in the window)

  name gene type chromosome strand annotation_start annotation_end has_transcript transcription_end transcription_start YEL072W RMD6 gene chrV 1 13720 14415 1 14745 13569 YEL071W DLD3 gene chrV 1 16355 17845 1 17881 16177 YEL070W DSF1 gene chrV 1 19589 21097 1 21197 19539

  craw_coverage --bam file.bam --annot annot.txt --ref-col annotation_start --start-col annotation_start --stop-col annotation_end

The position of reference must be between start and end. The authorized values are positive integers.

The position of reference can be used to define the reference and the start ot the end of the window.

 craw_coverage --bam file.bam --annot annot.txt --ref-col annotation_start --start-col annotation_start --stop-col annotation_end

All other columns are not necessary but will be reported as is in the coverage file.

Outputs

coverage_file

It's a tsv file with all columns found in annotation file plus the result of coverage position by position centered on the reference position define for each line. for instance

craw_coverage -bam=../data/craw_data_test/WTE1.bam --annot=../data/craw_data_test/annotations.txt
--ref-col=annotation_start --before=0  --after=2000

In the command line above, the column '0' correspond to the annotation_start position the column '1' to annotation_start + 1 on so on until '2000' (here we display only the first 3 columns of the coverage).

# Running Counter RnAseq Window
# Version: craw NOT packaged, it should be a development version | Python 3.4
# With the following arguments:
# --after=2000
# --annot=../data/craw_data_test/annotations.txt
# --bam=../data/craw_data_test/WTE1.bam
# --before=0
# --output=WTE1_0+2000.new.cov
# --qual-thr=15
# --ref-col=annotation_start
# --suffix=cov
sense   name    gene    type    chromosome      strand  annotation_start        annotation_end  has_transcript  transcription_end       transcription_start     0       1       2
S       YEL072W RMD6    gene    chrV    +       13720   14415   1       14745   13569   7       7       7
AS      YEL072W RMD6    gene    chrV    +       13720   14415   1       14745   13569   0       0       0
S       YEL071W DLD3    gene    chrV    +       16355   17845   1       17881   16177   31      33      33

The line starting with '#' are comments and will be ignored for further processing. But in traceability/reproducibility concern, in the comments craw_coverage indicate the version of the program and the arguments used for this experiment.

craw_htmp

Inputs

see craw_coverage output

Outputs

The default output of craw_htmp (if --out is omitted) is graphical window on the screen. The figure display on the screen can be saved using the window menu. It is also possible to generate directly a image file in various format by specifying the --out option. The output format will be deduced form the filename extension provide to --out option.

--out foo.jpeg for jpeg image or --out foo.png for png image

The supported format vary in function of the matplotlib backend used (see ).

If --size raw is used 2 files will be generated one for the sense and the other for the antisense. If --out is not specified it will be the name of the coverage file without extension and the format will be png.

craw_htmp foo_bar.cov --size raw

will produce foo_bar.sense.png and foo_bar.antisense.png

craw_htmp foo_bar.cov --size raw --out Xyzzy.jpeg

will produce Xyzzy.sense.jpeg and Xyzzy.antisense.jpeg

Command line options

There is many options for each craw scripts to have an exhaustive list of options use --help option or read the manual (html or pdf)