Counter RNA seq Window is a package which aim to compute and visualize the coverage of RNA seq experiment.
- python > 3
- psutil >= 4.0
- pysam >= 0.9.1.4
- pandas >= 0.17.1
- numpy >= 1.11.2
- matplotlib >= 1.5.3
- pillow >= 3.4.2
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/
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
You can also use the package without install it. You have to export the CRAW_HOME environment variable. Then you can use it directly
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 -vvv
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)
A detailed documentation is available
- online: latest documentation for master
- installed along craw in INSTALL_DIR/share/craw/doc/(html|pdf)
Inputs / Outputs
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/ .
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.
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
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.
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.
see craw_coverage output
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 option for each craw scripts to have an exhausted list of options use –help options or read the manual (html or pdf)