.. _inputs-outputs: ================ Inputs / Outputs ================ craw_coverage ============= Inputs ------ craw_coverage* need a file bam or wig to compute coverage and an annotation file to specify on which regions to compute these coverages. bam file ^^^^^^^^ *craw_coverage* can use 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. annotation file ^^^^^^^^^^^^^^^ The annotation file is a `tsv` file by default. It's mean that it is a text file with value separated by tabulation (not spaces) or commas. But if a separator is specified (--sep) it can be a csv file or any columns file. 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 YEL066W HPA3 chrV + 27206 YEL065W SIT1 chrV + 29543 YEL062W NPR2 chrV + 36254 YEL058W PCM1 chrV + 44925 YEL056W HAT2 chrV + 48373 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 YEL066W HPA3 chrV + 27206 YEL065W SIT1 chrV + 29543 YEL062W NPR2 chrV + 36254 YEL058W PCM1 chrV + 44925 YEL056W HAT2 chrV + 48373 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 YEL066W HPA3 gene chrV 1 26721 27206 1 27625 26137 YEL065W SIT1 gene chrV 1 27657 29543 1 29601 27625 YEL062W NPR2 gene chrV 1 34407 36254 1 36401 34321 YEL058W PCM1 gene chrV 1 43252 44925 1 44993 43217 YEL056W HAT2 gene chrV 1 47168 48373 1 48457 47105 YEL052W AFG1 gene chrV 1 56571 58100 1 58105 56537 :: craw_coverage --wig file.wig --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. .. note:: 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. .. _cov_out: 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 --wig=../data/small.wig --annot=../data/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 craw_coverage # Version: craw NOT packaged, it should be a development version | Python 3.4 # Using: pysam 0.9.1.4 (samtools 1.3.1) # # craw_coverage run with the following arguments: # --after=3 # --annot=../data/annotation_wo_start.txt # --before=5 # --chr-col=chromosome # --output=small_wig.cov # --qual-thr=0 # --quiet=1 # --ref-col=Position # --sense=mixed # --sep= # --strand-col=strand # --suffix=cov # --verbose=0 # --wig=../data/small.wig 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 `cov_out`_ Outputs ------- The default output of *craw_htmp* (if --out is omitted) is grapical window on the screen. The figure display on the screen can be saved using the window menu. .. container:: .. image:: _static/craw_htmp_sense_on_left.png :width: 30% :alt: --sense-on-left 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 matloblib backend used (see :ref:`matplotlibrc`). 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*