Mercurial > repos > biocomp-ibens > alfa
changeset 0:e360f840a92e draft default tip
Uploaded
| author | biocomp-ibens |
|---|---|
| date | Wed, 16 May 2018 09:49:18 -0400 |
| parents | |
| children | |
| files | alfa/.shed.yml alfa/ALFA.py alfa/ALFA.xml alfa/ALFA_wrapper.py alfa/test-data/alfa_toy-Biofeatures Distribution.pdf alfa/test-data/alfa_toy.bam alfa/test-data/alfa_toy.bedgraph alfa/test-data/alfa_toy.categories_counts alfa/test-data/alfa_toy.gtf alfa/test-data/alfa_toy.stranded.index alfa/test-data/alfa_toy.unstranded.index alfa/tool-data/alfa_indexes.loc.sample alfa/tool_data_table_conf.xml.sample alfa/tool_dependencies.xml |
| diffstat | 14 files changed, 2441 insertions(+), 0 deletions(-) [+] |
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/.shed.yml Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,14 @@ +categories: +- Graphics +- Next Gen Mappers +- Sequence Analysis +- Visualization +description: Plot the distribution of the genomic features captured by aligned reads +long_description: + ALFA provides a global overview of features distribution composing New Generation Sequencing dataset(s). + Given a set of aligned reads (BAM files) and an annotation file (GTF format), the tool produces plots of the raw and normalized distributions of those reads among genomic categories (stop codon, 5'-UTR, CDS, intergenic, etc.) and biotypes (protein coding genes, miRNA, tRNA, etc.). Whatever the sequencing technique, whatever the organism. + https://github.com/biocompibens/ALFA +name: alfa +owner: charles_bernard +remote_repository_url: https://github.com/biocompibens/ALFA/tree/master/Galaxy_toolshed_repositories/ALFA +type: unrestricted
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/ALFA.py Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,1850 @@ +#!/usr/bin/python +# -*- coding: utf-8 -*- + +__author__ = "noel & bahin" +""" ALFA provides a global overview of features distribution composing NGS dataset(s). """ + +import argparse +import os +import numpy +import copy +import sys +import subprocess +import matplotlib.pyplot as plt +import matplotlib.cm as cmx +import matplotlib.colors as colors +import matplotlib.patheffects as PathEffects +import re +from matplotlib.backends.backend_pdf import PdfPages +# To correctly embed the texts when saving plots in svg format +import matplotlib +# import progressbar +import collections +import matplotlib as mpl +import numpy as np + +matplotlib.rcParams["svg.fonttype"] = "none" + + +########################################################################## +# FUNCTIONS # +########################################################################## + +def init_dict(d, key, init): + if key not in d: + d[key] = init + + +def tryint(s): + """ Function called by "alphanum_key" function to sort the chromosome names. """ + try: + return int(s) + except ValueError: + return s + + +def alphanum_key(s): + """ Turn a string into a list of string and number chunks. + "z23a" -> ["z", 23, "a"] + """ + return [ tryint(c) for c in re.split("([0-9]+)", s) ] + + +def required_arg(arg, aliases): + """ Function to display the help and quit if a required argument is missing. """ + if not arg: + print >> sys.stderr, "\nError: %s argument is missing.\n" % aliases + parser.print_usage() + sys.exit(1) + + +def existing_file(filename): + """ Checks if filename already exists and exit if so. """ + if os.path.isfile(filename): + sys.exit("Error: The file '" + filename + "' is about to be produced but already exists in the directory. \n### End of program") + + +def get_chromosome_lengths(): + """ + Parse the file containing the chromosome lengths. + If no length file is provided, browse the annotation file (GTF) to estimate the chromosome sizes. + """ + lengths = {} + gtf_chrom_names = set() + # If the user provided a chromosome length file + if options.chr_len: + # Getting the chromosome lengths from the chromosome lengths file + with open(options.chr_len, "r") as chr_len_fh: + for line in chr_len_fh: + try: + lengths[line.split("\t")[0]] = int(line.rstrip().split("\t")[1]) + except IndexError: + sys.exit("Error: The chromosome lengths file is not correctly formed. It is supposed to be tabulated file with two fields per line.") + # Getting the chromosome lengths from the GTF file + with open(options.annotation, "r") as gtf_fh: + for line in gtf_fh: + if not line.startswith("#"): + gtf_chrom_names.add(line.split("\t")[0]) + # Checking if the chromosomes from the chromosome lengths file are present in the GTF file + for chrom in lengths: + if chrom not in gtf_chrom_names: + print >> sys.stderr, "Warning: chromosome '" + chrom + "' of the chromosome lengths file does not match any chromosome name in the GTF file provided and was ignored." + # Checking if the chromosomes from the GTF file are present in the lengths file + for chrom in gtf_chrom_names: + if chrom not in lengths: + print >> sys.stderr, "Warning: at least one chromosome ('" + chrom + "') was found in the GTF file and does not match any chromosome provided in the lengths file." + print >> sys.stderr, "\t=> All the chromosome lengths will be approximated using annotations in the GTF file." + break + else: + return lengths + # If no chromosome lengths file was provided or if at least one chromosome was missing in the file, the end of the last annotation of the chromosome in the GTF file is considered as the chromosome length + with open(options.annotation, "r") as gtf_fh: + for line in gtf_fh: + if not line.startswith("#"): + chrom = line.split("\t")[0] + end = int(line.split("\t")[4]) + init_dict(lengths, chrom, 0) + lengths[chrom] = max(lengths[chrom], end) + print "The chromosome lengths have been approximated using the GTF file annotations (the stop position of the last annotation of each chromosome is considered as its length)." + return lengths + + +def write_feature_on_index(feat, chrom, start, stop, sign, stranded_genome_index): + """ Write one new line in the stranded index file and, if necessary, the unstranded index file. """ + grouped_by_biotype_features = [] + for biotype, categs in feat.iteritems(): + categ_list = [] + for cat in set(categs): + categ_list.append(cat) + grouped_by_biotype_features.append(":".join((str(biotype), ",".join(categ_list)))) + + #stranded_genome_index.write('\t'.join((chrom, start, stop, sign, '')) + '\t'.join(grouped_by_biotype_features) + '\n') + stranded_genome_index.write( + '\t'.join((chrom, start, stop, sign)) + '\t' + '\t'.join(grouped_by_biotype_features) + '\n') + if unstranded_genome_index: ## MB: Why? Not always unstranded and stranded?? + #unstranded_genome_index.write('\t'.join((chrom, start, stop, '.', '')) + '\t'.join(grouped_by_biotype_features) + '\n') + unstranded_genome_index.write( + '\t'.join((chrom, start, stop, '.')) + '\t' + '\t'.join(grouped_by_biotype_features) + '\n') + + +def write_index_line(feat, chrom, start, stop, sign, fh): + """ Write a new line in an index file. """ + # Formatting the features info + feat_by_biotype = [] + for biot, cat in feat.iteritems(): + #feat_by_biotype.append(":".join((str(biot), ",".join(set(cat))))) + feat_by_biotype.append(":".join((str(biot), ",".join(set(cat))))) + # Writing the features info in the index file + fh.write("\t".join((chrom, start, stop, sign)) + "\t" + "\t".join(feat_by_biotype) + "\n") + + +def write_index(feat_values, chrom, start, stop, stranded_genome_index, unstranded_genome_index): + """ Writing the features info in the proper index files. """ + # Writing info to the stranded indexes + if feat_values[0] != {}: + write_index_line(feat_values[0], chrom, start, stop, "+", stranded_genome_index) + else: + stranded_genome_index.write("\t".join((chrom, start, stop, "+", "antisense\n"))) + if feat_values[1] != {}: + write_index_line(feat_values[1], chrom, start, stop, "-", stranded_genome_index) + else: + stranded_genome_index.write("\t".join((chrom, start, stop, "-", "antisense\n"))) + # Writing info to the unstranded index + unstranded_feat = dict(feat_values[0], **feat_values[1]) + for name in set(feat_values[0]) & set(feat_values[1]): + unstranded_feat[name] += feat_values[0][name] + write_index_line(unstranded_feat, chrom, start, stop, ".", unstranded_genome_index) + + +def count_genome_features(cpt, features, start, stop, coverage=1): + """ Reads genome index and registers feature counts. """ + # If no biotype priority: category with the highest priority for each found biotype has the same weight (1/n_biotypes) + if not biotype_prios: + nb_biot = len(features) + # For each categ(s)/biotype pairs + for feat in features: + cur_prio = 0 + # Separate categorie(s) and biotype + try: + biot, cats = feat.split(":") + # Error if the feature is "antisense": update the "antisense/antisense" counts + except ValueError: + try: + cpt[("antisense", "antisense")] += (int(stop) - int(start)) * coverage / float(nb_biot) + except KeyError: + cpt[("antisense", "antisense")] = (int(stop) - int(start)) * coverage / float(nb_biot) + return None + # Browse the categories and get only the one(s) with highest priority + for cat in cats.split(","): + try: + prio = prios[cat] + except KeyError: + # TODO: Find a way to add unknown categories + if cat not in unknown_cat: + print >> sys.stderr, "Warning: Unknown categorie '%s' found and ignored.\n" % cat, + unknown_cat.add(cat) + continue + # Check if the category has a highest priority than the current one + if prio > cur_prio: + cur_prio = prio + cur_cat = {cat} + if prio == cur_prio: + cur_cat.add(cat) + # Increase each counts by the coverage divided by the number of categories and biotypes + nb_cat = len(cur_cat) + for cat in cur_cat: + try: + cpt[(cat, biot)] += (int(stop) - int(start)) * coverage / (float(nb_biot * nb_cat)) + except KeyError: + cpt[(cat, biot)] = (int(stop) - int(start)) * coverage / (float(nb_biot * nb_cat)) + else: + # TODO: Add an option to provide biotype priorities and handle it + pass + +''' +def add_info(cpt, feat_values, start, stop, chrom=None, unstranded_genome_index=None, stranded_genome_index=None, + biotype_prios=None, coverage=1, categ_prios=None): + """ + From an annotated genomic interval (start/stop positions and associated feature: one or more category(ies)/biotype pair(s) ) + - If a file is provided: write the information at the end of the index file being generated; + - else: browse the features and update the counts of categories/biotypes found in the genome. + """ + ## Writing in the file if it was provided + if stranded_genome_index: + # If only one strand has a feature, this feature will directly be written on the unstranded index + if feat_values[0] == {}: + stranded_genome_index.write('\t'.join((chrom, start, stop, '+', 'antisense\n'))) + elif feat_values[1] == {}: + stranded_genome_index.write('\t'.join((chrom, start, stop, '-', 'antisense\n'))) + + write_feature_on_index(feat_values[0], chrom, start, stop, '+', stranded_genome_index) + write_feature_on_index(feat_values[1], chrom, start, stop, '-', stranded_genome_index) + unstranded_feat = dict(feat_values[0], **feat_values[1]) + for name in set(feat_values[0]) & set(feat_values[1]): + unstranded_feat[name] += feat_values[0][name] + write_feature_on_index(unstranded_feat, chrom, start, stop, '.', unstranded_genome_index) + + if feat_values[0] == {}: + # An interval with no feature corresponds to a region annotated only on the reverse strand: update 'antisense' counts + stranded_genome_index.write('\t'.join((chrom, start, stop, '+', 'antisense\n'))) + #write_feature_on_index(feat_values[1], chrom, start, stop, '-', stranded_genome_index, unstranded_genome_index) + write_feature_on_index(feat_values[1], chrom, start, stop, '-', stranded_genome_index) + write_feature_on_index(feat_values[1], chrom, start, stop, '.', unstranded_genome_index) + elif feat_values[1] == {}: + #write_feature_on_index(feat_values[0], chrom, start, stop, '+', stranded_genome_index, unstranded_genome_index) + write_feature_on_index(feat_values[0], chrom, start, stop, '+', stranded_genome_index) + write_feature_on_index(feat_values[0], chrom, start, stop, '.', unstranded_genome_index) + stranded_genome_index.write('\t'.join((chrom, start, stop, '-', 'antisense\n'))) + # Else, the unstranded index should contain the union of plus and minus features + else: + write_feature_on_index(feat_values[0], chrom, start, stop, '+', stranded_genome_index) + write_feature_on_index(feat_values[1], chrom, start, stop, '-', stranded_genome_index) + unstranded_feat = dict(feat_values[0], **feat_values[1]) + for name in set(feat_values[0]) & set(feat_values[1]): + unstranded_feat[name] += feat_values[0][name] + write_feature_on_index(unstranded_feat, chrom, start, stop, '.', unstranded_genome_index) + + ## Increasing category counter(s) + else: ##MB Why increase if file not provided?? + # Default behavior if no biotype priorities : category with the highest priority for each found biotype has the same weight (1/n_biotypes) + if not biotype_prios: + nb_feat = len(feat_values) ## MB: nb biotypes?? + # For every categ(s)/biotype pairs + for feat in feat_values: ## MB: for each biotype + cur_prio = 0 + selected_categ = [] + # Separate categorie(s) and biotype + try: + biot, cats = feat.split(":") + # Error if feature equal "antisense" : update the 'antisense/antisense' counts + except ValueError: + try: + cpt[(feat, feat)] += (int(stop) - int(start)) * coverage / float(nb_feat) ## MB: clearly write 'antisense'?? + except: ## MB: add a KeyError exception?? + cpt[(feat, feat)] = (int(stop) - int(start)) * coverage / float(nb_feat) + return + # Browse the categories and get only the one(s) with highest priority + for cat in cats.split(','): + try: + prio = prios[cat] + except: ## MB: KeyError?? + # TODO Find a way to add unknown categories + if cat not in unknown_feature: + print >> sys.stderr, "Warning: Unknown categorie %s found and ignored.\n" % cat, + unknown_feature.append(cat) + continue + if prio > cur_prio: + cur_prio = prio + selected_categ = [cat] + if prio == cur_prio: + if cat != selected_categ: ## MB: work with set?? + try: + if cat not in selected_categ: + selected_categ.append(cat) + except TypeError: ## What TypeError?? + selected_categ = [selected_categ, cat] + # Increase each counts by the coverage divided by the number of categories and biotypes + nb_cats = len(selected_categ) + for cat in selected_categ: + try: + cpt[(cat, biot)] += (int(stop) - int(start)) * coverage / (float(nb_feat * nb_cats)) + except KeyError: + cpt[(cat, biot)] = (int(stop) - int(start)) * coverage / (float(nb_feat * nb_cats)) + # else : + # cpt[(cats,biot)] = (int(stop) - int(start)) / float(nb_feat) * coverage + # Else, apply biotype selection according to the priority set + else: + # TODO Add an option to pass biotype priorities and handle it + pass +''' + +def register_interval(features_dict, chrom, stranded_index_fh, unstranded_index_fh): + """ Write the interval features info into the genome index files. """ + # Adding the chromosome to the list if not present + if chrom not in index_chrom_list: + index_chrom_list.append(chrom) + # Writing the chromosome in the index file + with open(unstranded_index_fh, "a") as unstranded_index_fh, open(stranded_index_fh, "a") as stranded_index_fh: + # Initializing the first interval start and features + sorted_pos = sorted(features_dict["+"].keys()) + interval_start = sorted_pos[0] + features_plus = features_dict["+"][interval_start] + features_minus = features_dict["-"][interval_start] + # Browsing the interval boundaries + for interval_stop in sorted_pos[1:]: + # Writing the current interval features to the indexes + write_index([features_plus, features_minus], chrom, str(interval_start), str(interval_stop), stranded_index_fh, unstranded_index_fh) + # Initializing the new interval start and features + interval_start = interval_stop + # Store current features + prev_features_plus = features_plus + prev_features_minus = features_minus + # Update features + features_plus = features_dict["+"][interval_start] + features_minus = features_dict["-"][interval_start] + # If feature == transcript and prev interval's feature is exon => add intron feature + for biotype, categ in features_plus.iteritems(): + if set(categ) == {"gene", "transcript"}: + if "exon" in prev_features_plus[biotype]: + categ.append("intron") + else: + continue + for biotype, categ in features_minus.iteritems(): + if set(categ) == {"gene", "transcript"}: + if "exon" in prev_features_minus[biotype]: + categ.append("intron") + else: + continue + + + +def generate_genome_index(annotation, unstranded_genome_index, stranded_genome_index, chrom_sizes): + """ Create an index of the genome annotations and save it in a file. """ + # Initializations + intervals_dict = {} + max_value = -1 + prev_chrom = "" + i = 0 # Line counter + # Write the chromosome lengths as comment lines before the genome index + with open(unstranded_genome_index, "w") as unstranded_index_fh, open(stranded_genome_index, "w") as stranded_index_fh: + for key, value in chrom_sizes.items(): + unstranded_index_fh.write("#%s\t%s\n" % (key, value)) + stranded_index_fh.write("#%s\t%s\n" % (key, value)) + # Progress bar to track the genome indexes creation + nb_lines = sum(1 for _ in open(annotation)) + # pbar = progressbar.ProgressBar(widgets=["Indexing the genome ", progressbar.Percentage(), " ", progressbar.Bar(), progressbar.Timer()], maxval=nb_lines).start() + # Browsing the GTF file and writing into genome index files + with open(annotation, "r") as gtf_fh: + for line in gtf_fh: + i += 1 + # Update the progressbar every 1k lines + # if i % 1000 == 1: + # pbar.update(i) + # Processing lines except comment ones + if not line.startswith("#"): + # Getting the line info + line_split = line.rstrip().split("\t") + chrom = line_split[0] + cat = line_split[2] + start = int(line_split[3]) - 1 + stop = int(line_split[4]) + strand = line_split[6] + antisense = reverse_strand[strand] + biotype = line_split[8].split("biotype")[1].split(";")[0].strip('" ') + # Registering stored features info in the genome index file(s) if the new line concerns a new chromosome or the new line concerns an annotation not overlapping previously recorded ones + if start > max_value or chrom != prev_chrom: + # Write the previous features + if intervals_dict: + register_interval(intervals_dict, prev_chrom, stranded_genome_index, unstranded_genome_index) + prev_chrom = chrom + # (Re)Initializing the intervals info dict + intervals_dict = {strand: {start: {biotype: [cat]}, stop: {}}, antisense: {start: {}, stop: {}}} + max_value = stop + + # Update the dictionary which represents intervals for every distinct annotation + else: + # Storing the intervals on the strand of the current feature + stranded_intervals = intervals_dict[strand] + added_info = False # Variable to know if the features info were already added + # Browsing the existing boundaries + for boundary in sorted(stranded_intervals): + # While the GTF line start is after the browsed boundary: keep the browsed boundary features info in case the GTF line start is before the next boundary + if boundary < start: + stored_feat_strand, stored_feat_antisense = [dict(stranded_intervals[boundary]), dict(intervals_dict[antisense][boundary])] + + # The GTF line start is already an existing boundary: store the existing features info (to manage after the GTF line stop) and update it with the GTF line features info + elif boundary == start: + stored_feat_strand, stored_feat_antisense = [dict(stranded_intervals[boundary]), dict(intervals_dict[antisense][boundary])] + # Adding the GTF line features info to the interval + try: + stranded_intervals[boundary][biotype] = stranded_intervals[boundary][biotype] + [cat] + except KeyError: # If the GTF line features info regard an unregistered biotype + stranded_intervals[boundary][biotype] = [cat] + added_info = True # The features info were added + + # The browsed boundary is after the GTF line start: add the GTF line features info + elif boundary > start: + # Create a new boundary for the GTF line start if necessary (if it is between 2 existing boundaries, it was not created before) + if not added_info: + stranded_intervals[start] = copy.deepcopy(stored_feat_strand) + #stranded_intervals[start][biotype] = [cat] + try: + stranded_intervals[start][biotype].append(cat) + except KeyError: + stranded_intervals[start][biotype] = [cat] + intervals_dict[antisense][start] = copy.deepcopy(stored_feat_antisense) + added_info = True # The features info were added + # While the browsed boundary is before the GTF line stop: store the existing features info (to manage after the GTF line stop) and update it with the GTF line features info + if boundary < stop: + stored_feat_strand, stored_feat_antisense = [dict(stranded_intervals[boundary]), dict(intervals_dict[antisense][boundary])] + try: + stranded_intervals[boundary][biotype] = stranded_intervals[boundary][biotype] + [cat] + except KeyError: + stranded_intervals[boundary][biotype] = [cat] + # The GTF line stop is already exists, nothing more to do, the GTF line features info are integrated + elif boundary == stop: + break + # The browsed boundary is after the GTF line stop: create a new boundary for the GTF line stop (with the stored features info) + else: # boundary > stop + stranded_intervals[stop] = copy.deepcopy(stored_feat_strand) + intervals_dict[antisense][stop] = copy.deepcopy(stored_feat_antisense) + break # The GTF line features info are integrated + # If the GTF line stop is after the last boundary, extend the dictionary + if stop > max_value: + max_value = stop + stranded_intervals[stop] = {} + intervals_dict[antisense][stop] = {} + + # Store the categories of the last chromosome + register_interval(intervals_dict, chrom, stranded_genome_index, unstranded_genome_index) + # pbar.finish() + + +def generate_bedgraph_files(sample_labels, bam_files): + """ Creates BedGraph files from BAM ones and return filenames and labels. """ + #sample_files = [] + #sample_labels = [] + # Progress bar to track the BedGraph file creation + #pbar = progressbar.ProgressBar(widgets=["Generating the BedGraph files ", progressbar.Percentage(), progressbar.Bar(), progressbar.Timer()], max_value=len(bam_files)+1).start() + # pbar = progressbar.ProgressBar(widgets=["Generating the BedGraph files ", progressbar.Percentage(), progressbar.Bar(), progressbar.Timer()], maxval=len(sample_labels)+1).start() + n = 1 + # pbar.update(n) + #for n in range(0, len(bam_files), 2): + for sample_label, bam_file in zip(sample_labels, bam_files): + # Get the label for this sample + #sample_labels.append(bam_files[n + 1]) + # Modify it to contain only alphanumeric characters (avoid files generation with dangerous names) + #modified_label = "_".join(re.findall(r"[\w']+", bam_files[n + 1])) + if options.strandness in ["forward", "fr-firststrand"]: + #subprocess.call("bedtools genomecov -bg -split -strand + -ibam " + bam_files[n] + " > " + modified_label + ".plus.bedgraph", shell=True) + subprocess.call("bedtools genomecov -bg -split -strand + -ibam " + bam_file + " > " + sample_label + ".plus.bedgraph", shell=True) + # pbar.update(n + 0.5) + #subprocess.call("bedtools genomecov -bg -split -strand - -ibam " + bam_files[n] + " > " + modified_label + ".minus.bedgraph", shell=True) + subprocess.call("bedtools genomecov -bg -split -strand - -ibam " + bam_file + " > " + sample_label + ".minus.bedgraph", shell=True) + # pbar.update(n + 0.5) + elif options.strandness in ["reverse", "fr-secondstrand"]: + #subprocess.call("bedtools genomecov -bg -split -strand - -ibam " + bam_files[n] + " > " + modified_label + ".plus.bedgraph", shell=True) + subprocess.call("bedtools genomecov -bg -split -strand - -ibam " + bam_file + " > " + sample_label + ".plus.bedgraph", shell=True) + # pbar.update(n + 0.5) + #subprocess.call("bedtools genomecov -bg -split -strand + -ibam " + bam_files[n] + " > " + modified_label + ".minus.bedgraph", shell=True) + subprocess.call("bedtools genomecov -bg -split -strand + -ibam " + bam_file + " > " + sample_label + ".minus.bedgraph", shell=True) + # pbar.update(n + 0.5) + else: + #subprocess.call("bedtools genomecov -bg -split -ibam " + bam_files[n] + " > " + modified_label + ".bedgraph", shell=True) + subprocess.call("bedtools genomecov -bg -split -ibam " + bam_file + " > " + sample_label + ".bedgraph", shell=True) + # pbar.update(n + 1) + #sample_files.append(modified_label) + # pbar.finish() + #return sample_files, sample_labels + return None + + +def read_gtf(gtf_index_file, sign): + global gtf_line, gtf_chrom, gtf_start, gtf_stop, gtf_features, endGTF + strand = "" + while strand != sign: + gtf_line = gtf_index_file.readline() + # If the GTF file is finished + if not gtf_line: + endGTF = True + return endGTF + splitline = gtf_line.rstrip().split("\t") + try: + strand = splitline[3] + # strand information can not be found in the file file header + except IndexError: + pass + gtf_chrom = splitline[0] + gtf_features = splitline[4:] + gtf_start, gtf_stop = map(int, splitline[1:3]) + return endGTF + + +#def read_counts(counts_files): +def read_counts(sample_labels, counts_files): + """ Reads the counts from an input file. """ + cpt = {} + cpt_genome = {} + #for fcounts in counts_files: + for sample_label, filename in zip(sample_labels, counts_files): + #label = os.path.splitext(os.path.basename(fcounts))[0] + #labels.append(label) + #cpt[label] = {} + cpt[sample_label] = {} + #with open(fcounts, "r") as counts_fh: + with open(filename, "r") as counts_fh: + for line in counts_fh: + if not line.startswith("#"): + feature = tuple(line.split("\t")[0].split(",")) + #cpt[label][feature] = float(line.split("\t")[1]) + cpt[sample_label][feature] = float(line.split("\t")[1]) + cpt_genome[feature] = float(line.rstrip().split("\t")[2]) + #return cpt, cpt_genome, labels + return cpt, cpt_genome + + +def get_chromosome_names_in_index(genome_index): + """ Returns the chromosome names in a genome index file. """ + with open(genome_index, "r") as index_fh: + for line in index_fh: + if not line.startswith("#") and (line.split("\t")[0] not in index_chrom_list): + index_chrom_list.append(line.split("\t")[0]) + return index_chrom_list + + +def read_index(): + """ Parse index files info (chromosomes list, lengths and genome features). """ + with open(genome_index, "r") as index_fh: + for line in index_fh: + if line.startswith("#"): + lengths[line.split("\t")[0][1:]] = int(line.split("\t")[1]) + else: + chrom = line.split("\t")[0] + if chrom not in index_chrom_list: + index_chrom_list.append(chrom) + count_genome_features(cpt_genome, line.rstrip().split("\t")[4:], line.split("\t")[1], line.split("\t")[2]) + + +#def intersect_bedgraphs_and_index_to_counts_categories(sample_files, sample_labels, prios, genome_index, biotype_prios=None): ## MB: To review +def intersect_bedgraphs_and_index_to_counts_categories(sample_labels, bedgraph_files, biotype_prios=None): ## MB: To review + global gtf_line, gtf_chrom, gtf_start, gtf_stop, gtf_cat, endGTF + unknown_chrom = [] + cpt = {} # Counter for the nucleotides in the BAM input file(s) + #for n in range(len(sample_files)): + if bedgraph_files == []: + bedgraph_files = sample_labels + for sample_label, bedgraph_basename in zip(sample_labels, bedgraph_files): + #sample_file = sample_files[n] + #sample_name = sample_labels[n] + # Initializing the category counter dict for this sample and the number of lines to process for the progress bar + #init_dict(cpt, sample_name, {}) + init_dict(cpt, sample_label, {}) + bg_extension = ".bedgraph" + if options.strandness == "unstranded": + strands = [("", ".")] + #nb_lines = sum(1 for _ in open(sample_file + ".bedgraph")) + try : + nb_lines = sum(1 for _ in open(bedgraph_basename + bg_extension)) + except IOError: + bg_extension = "bg" + nb_lines = sum(1 for _ in open(bedgraph_basename + bg_extension)) + else: + strands = [(".plus", "+"), (".minus", "-")] + #nb_lines = sum(1 for _ in open(sample_file + ".plus.bedgraph")) + sum(1 for _ in open(sample_file + ".minus.bedgraph")) + try: + nb_lines = sum(1 for _ in open(bedgraph_basename + ".plus" + bg_extension)) + sum(1 for _ in open(bedgraph_basename + ".minus" + bg_extension)) + except IOError: + nb_lines = sum(1 for _ in open(bedgraph_basename + ".plus" + bg_extension)) + sum( + 1 for _ in open(bedgraph_basename + ".minus" + bg_extension)) + + # Progress bar to track the BedGraph and index intersection + #pbar = progressbar.ProgressBar(widgets=["Processing " + sample_file + " ", progressbar.Percentage(), progressbar.Bar(), progressbar.Timer()], max_value=nb_lines).start() + # pbar = progressbar.ProgressBar(widgets=["Processing " + sample_label + " ", progressbar.Percentage(), progressbar.Bar(), progressbar.Timer()], maxval=nb_lines).start() + i = 0 + + # Intersecting the BedGraph and index files + for strand, sign in strands: + prev_chrom = "" + endGTF = False # Reaching the next chr or the end of the GTF index + intergenic_adds = 0.0 + #with open(sample_file + strand + ".bedgraph", "r") as bedgraph_fh: + with open(bedgraph_basename + strand + bg_extension, "r") as bedgraph_fh: + # Running through the BedGraph file + for bam_line in bedgraph_fh: + i += 1 + # if i % 10000 == 0: + # pbar.update(i) + # Getting the BAM line info + bam_chrom = bam_line.split("\t")[0] + bam_start, bam_stop, bam_cpt = map(float, bam_line.split("\t")[1:4]) + # Skip the line if the chromosome is not in the index + if bam_chrom not in index_chrom_list: + if bam_chrom not in unknown_chrom: + unknown_chrom.append(bam_chrom) + print "\r \r Chromosome '" + bam_chrom + "' not found in index." # MB: to adapt with the progress bar + continue + # If this is a new chromosome (or the first one) + if bam_chrom != prev_chrom: + intergenic_adds = 0.0 + # (Re)opening the GTF index and looking for the first line of the matching chr + try: + gtf_index_file.close() + except UnboundLocalError: + pass + gtf_index_file = open(genome_index, "r") + endGTF = False + read_gtf(gtf_index_file, sign) + while bam_chrom != gtf_chrom: + read_gtf(gtf_index_file, sign) + if endGTF: + break + prev_chrom = bam_chrom + + # Looking for the first matching annotation in the GTF index + while (not endGTF) and (gtf_chrom == bam_chrom) and (bam_start >= gtf_stop): + read_gtf(gtf_index_file, sign) + if gtf_chrom != bam_chrom: + endGTF = True + # Processing BAM lines before the first GTF annotation if there are + if bam_start < gtf_start: + # Increase the "intergenic" category counter with all or part of the BAM interval + try: + intergenic_adds += min(bam_stop, gtf_start) - bam_start + #cpt[sample_name][("intergenic", "intergenic")] += (min(bam_stop, + cpt[sample_label][("intergenic", "intergenic")] += (min(bam_stop, + gtf_start) - bam_start) * bam_cpt + except KeyError: + #cpt[sample_name][("intergenic", "intergenic")] = (min(bam_stop, + cpt[sample_label][("intergenic", "intergenic")] = (min(bam_stop, + gtf_start) - bam_start) * bam_cpt + # Go to next line if the BAM interval was totally upstream the first GTF annotation, carry on with the remaining part otherwise + if endGTF or (bam_stop <= gtf_start): + continue + else: + bam_start = gtf_start + + # We can start the crossover + while not endGTF: + # Update category counter + #add_info(cpt[sample_name], gtf_features, bam_start, min(bam_stop, gtf_stop), coverage=bam_cpt) + #count_genome_features(cpt[sample_name], gtf_features, bam_start, min(bam_stop, gtf_stop), coverage=bam_cpt) + count_genome_features(cpt[sample_label], gtf_features, bam_start, min(bam_stop, gtf_stop), coverage=bam_cpt) + # Read the next GTF file line if the BAM line is not entirely covered + if bam_stop > gtf_stop: + # Update the BAM start pointer + bam_start = gtf_stop + endGTF = read_gtf(gtf_index_file, sign) + # If we read a new chromosome in the GTF file then it is considered finished + if bam_chrom != gtf_chrom: + endGTF = True + if endGTF: + break + else: + # Next if the BAM line is entirely covered + bam_start = bam_stop + break + + # Processing the end of the BAM line if necessary + if endGTF and (bam_stop > bam_start): + try: + #cpt[sample_name][("intergenic", "intergenic")] += (bam_stop - bam_start) * bam_cpt + cpt[sample_label][("intergenic", "intergenic")] += (bam_stop - bam_start) * bam_cpt + except KeyError: + #cpt[sample_name][("intergenic", "intergenic")] = (bam_stop - bam_start) * bam_cpt + cpt[sample_label][("intergenic", "intergenic")] = (bam_stop - bam_start) * bam_cpt + gtf_index_file.close() + # pbar.finish() + return cpt + + +def write_counts_in_files(cpt, genome_counts): + """ Writes the biotype/category counts in an output file. """ + for sample_label, counters in cpt.items(): + sample_label = "_".join(re.findall(r"[\w\-']+", sample_label)) + with open(sample_label + ".feature_counts.tsv", "w") as output_fh: + output_fh.write("#Category,biotype\tCounts_in_bam\tSize_in_genome\n") + for features_pair, counts in counters.items(): + output_fh.write("%s\t%s\t%s\n" % (",".join(features_pair), counts, genome_counts[features_pair])) + + +def recategorize_the_counts(cpt, cpt_genome, final): + final_cat_cpt = {} + final_genome_cpt = {} + for f in cpt: + # print "\nFinal categories for",f,"sample" + final_cat_cpt[f] = {} + for final_cat in final: + tot = 0 + tot_genome = 0 + for cat in final[final_cat]: + tot += cpt[f][cat] + tot_genome += cpt_genome[cat] + # output_file.write('\t'.join((final_cat, str(tot))) + '\n') + # print '\t'.join((final_cat, str(tot))) + final_cat_cpt[f][final_cat] = tot + final_genome_cpt[final_cat] = tot_genome + return final_cat_cpt, final_genome_cpt + + +def group_counts_by_categ(cpt, cpt_genome, final, selected_biotype): + final_cat_cpt = {} + final_genome_cpt = {} + filtered_cat_cpt = {} + for f in cpt: + final_cat_cpt[f] = {} + filtered_cat_cpt[f] = {} + for final_cat in final: + tot = 0 + tot_filter = 0 + tot_genome = 0 + for cat in final[final_cat]: + for key, value in cpt[f].items(): + if key[0] == cat: + tot += value + tot_genome += cpt_genome[key] + if key[1] == selected_biotype: + tot_filter += value + # output_file.write('\t'.join((final_cat, str(tot))) + '\n') + # print '\t'.join((final_cat, str(tot))) + final_cat_cpt[f][final_cat] = tot + if tot_genome == 0: + final_genome_cpt[final_cat] = 1e-100 + else: + final_genome_cpt[final_cat] = tot_genome + filtered_cat_cpt[f][final_cat] = tot_filter + # if "antisense" in final_genome_cpt: final_genome_cpt["antisense"] = 0 + return final_cat_cpt, final_genome_cpt, filtered_cat_cpt + + +def group_counts_by_biotype(cpt, cpt_genome, biotypes): + final_cpt = {} + final_genome_cpt = {} + for f in cpt: + final_cpt[f] = {} + for biot in biotypes: + tot = 0 + tot_genome = 0 + try: + for final_biot in biotypes[biot]: + for key, value in cpt[f].items(): + if key[1] == final_biot: + tot += value + # if key[1] != 'antisense': + tot_genome += cpt_genome[key] + except: + for key, value in cpt[f].items(): + if key[1] == biot: + tot += value + tot_genome += cpt_genome[key] + if tot != 0: + final_cpt[f][biot] = tot + final_genome_cpt[biot] = tot_genome + return final_cpt, final_genome_cpt + + +# def get_cmap(N): +# '''Returns a function that maps each index in 0, 1, ... N-1 to a distinct +# RGB color.''' +# color_norm = colors.Normalize(vmin=0, vmax=N-1) +# scalar_map = cmx.ScalarMappable(norm=color_norm, cmap='hsv') +# def map_index_to_rgb_color(index): +# return scalar_map.to_rgba(index) +# return map_index_to_rgb_color + +def one_sample_plot(ordered_categs, percentages, enrichment, n_cat, index, index_enrichment, bar_width, counts_type, + title, sample_labels): + ### Initialization + fig = plt.figure(figsize=(13, 9)) + ax1 = plt.subplot2grid((2, 4), (0, 0), colspan=2) + ax2 = plt.subplot2grid((2, 4), (1, 0), colspan=2) + cmap = plt.get_cmap("Spectral") + cols = [cmap(x) for x in xrange(0, 256, 256 / n_cat)] + if title: + ax1.set_title(title + "in: %s" % sample_labels[0]) + else: + ax1.set_title(counts_type + " distribution in mapped reads in: %s" % sample_labels[0]) + ax2.set_title("Normalized counts of " + counts_type) + + ### Barplots + # First barplot: percentage of reads in each categorie + ax1.bar(index, percentages, bar_width, + color=cols) + # Second barplot: enrichment relative to the genome for each categ + # (the reads count in a categ is divided by the categ size in the genome) + ax2.bar(index_enrichment, enrichment, bar_width, + color=cols, ) + ### Piecharts + pielabels = [ordered_categs[i] if percentages[i] > 0.025 else "" for i in xrange(n_cat)] + sum_enrichment = numpy.sum(enrichment) + pielabels_enrichment = [ordered_categs[i] if enrichment[i] / sum_enrichment > 0.025 else "" for i in xrange(n_cat)] + # Categories piechart + ax3 = plt.subplot2grid((2, 4), (0, 2)) + pie_wedge_collection, texts = ax3.pie(percentages, labels=pielabels, shadow=True, colors=cols) + # Enrichment piechart + ax4 = plt.subplot2grid((2, 4), (1, 2)) + pie_wedge_collection, texts = ax4.pie(enrichment, labels=pielabels_enrichment, shadow=True, colors=cols) + # Add legends (append percentages to labels) + labels = [" ".join((ordered_categs[i], "({:.1%})".format(percentages[i]))) for i in range(len(ordered_categs))] + ax3.legend(pie_wedge_collection, labels, loc="center", fancybox=True, shadow=True, prop={"size": "medium"}, + bbox_to_anchor=(1.7, 0.5)) + labels = [" ".join((ordered_categs[i], "({:.1%})".format(enrichment[i] / sum_enrichment))) for i in + range(len(ordered_categs))] # if ordered_categs[i] != "antisense"] + ax4.legend(pie_wedge_collection, labels, loc="center", fancybox=True, shadow=True, prop={"size": "medium"}, + bbox_to_anchor=(1.7, 0.5)) + # Set aspect ratio to be equal so that pie is drawn as a circle + ax3.set_aspect("equal") + ax4.set_aspect("equal") + return fig, ax1, ax2 + + +#def make_plot(ordered_categs, sample_names, categ_counts, genome_counts, pdf, counts_type, threshold, title=None, +def make_plot(sample_labels, ordered_categs, categ_counts, genome_counts, pdf, counts_type, threshold, title=None, svg=None, png=None, + categ_groups=[]): # MB: to review + # From ordered_categs, keep only the features (categs or biotypes) that we can find in at least one sample. + existing_categs = set() + for sample in categ_counts.values(): + existing_categs |= set(sample.keys()) + ordered_categs = filter(existing_categs.__contains__, ordered_categs) + xlabels = [cat if len(cat.split("_")) == 1 else "\n".join(cat.split("_")) if cat.split("_")[0] != 'undescribed' else "\n".join(["und.",cat.split("_")[1]]) for cat in ordered_categs] + n_cat = len(ordered_categs) + #n_exp = len(sample_names) + nb_samples = len(categ_counts) + ##Initialization of the matrix of counts (nrow=nb_experiements, ncol=nb_categorie) + #counts = numpy.matrix(numpy.zeros(shape=(n_exp, n_cat))) + counts = numpy.matrix(numpy.zeros(shape=(nb_samples, n_cat))) + ''' + for exp in xrange(len(sample_names)): + for cat in xrange(len(ordered_categs)): + try: + counts[exp, cat] = categ_counts[sample_names[exp]][ordered_categs[cat]] + except: + pass + ''' + for sample_label in sample_labels: + for cat in xrange(len(ordered_categs)): + try: + counts[sample_labels.index(sample_label), cat] = categ_counts[sample_label][ordered_categs[cat]] + except: + pass + + ##Normalize the categorie sizes by the total size to get percentages + sizes = [] + sizes_sum = 0 + for cat in ordered_categs: + sizes.append(genome_counts[cat]) + sizes_sum += genome_counts[cat] + if "antisense" in ordered_categs: + antisense_pos = ordered_categs.index("antisense") + sizes[antisense_pos] = 1e-100 + for cpt in xrange(len(sizes)): + sizes[cpt] /= float(sizes_sum) + + ## Create array which contains the percentage of reads in each categ for every sample + percentages = numpy.array(counts / numpy.sum(counts, axis=1)) + ## Create the enrichment array (counts divided by the categorie sizes in the genome) + enrichment = numpy.array(percentages / sizes) + if "antisense_pos" in locals(): + ''' + for i in xrange(len(sample_names)): + enrichment[i][antisense_pos] = 0 + ''' + for n in xrange(nb_samples): + enrichment[n][antisense_pos] = 0 + + # enrichment=numpy.log(numpy.array(percentages/sizes)) + #for exp in xrange(n_exp): + for n in xrange(nb_samples): + for i in xrange(n_cat): + val = enrichment[n][i] + if val > 1: + enrichment[n][i] = val - 1 + elif val == 1 or val == 0: + enrichment[n][i] = 0 + else: + enrichment[n][i] = -1 / val + 1 + + #### Finally, produce the plot + + ##Get the colors from the colormap + ncolor = 16 + cmap = ["#e47878", "#68b4e5", "#a3ea9b", "#ea9cf3", "#e5c957", "#a3ecd1", "#e97ca0", "#66d985", "#8e7ae5", + "#b3e04b", "#b884e4", "#e4e758", "#738ee3", "#e76688", "#70dddd", "#e49261"] + ''' + if n_exp > ncolor: + cmap = plt.get_cmap("Set3", n_exp) + cmap = [cmap(i) for i in xrange(n_exp)] + ''' + if nb_samples > ncolor: + cmap = plt.get_cmap("Set3", nb_samples) + cmap = [cmap(i) for i in xrange(nb_samples)] + + ## Parameters for the plot + opacity = 1 + # Create a vector which contains the position of each bar + index = numpy.arange(n_cat) + # Size of the bars (depends on the categs number) + #bar_width = 0.9 / n_exp + bar_width = 0.9 / nb_samples + + ##Initialise the subplot + # if there is only one sample, also plot piecharts + # if n_exp == 1 and counts_type.lower() == 'categories': + # fig, ax1, ax2 = one_sample_plot(ordered_categs, percentages[0], enrichment[0], n_cat, index, bar_width, counts_type, title) + ## If more than one sample + # else: + if counts_type.lower() != "categories": + #fig, (ax1, ax2) = plt.subplots(2, figsize=(5 + (n_cat + 2 * n_exp) / 3, 10)) + fig, (ax1, ax2) = plt.subplots(2, figsize=(5 + (n_cat + 2 * nb_samples) / 3, 10)) + else: + #fig, (ax1, ax2) = plt.subplots(2, figsize=(5 + (n_cat + 2 * n_exp) / 3, 10)) + fig, (ax1, ax2) = plt.subplots(2, figsize=(5 + (n_cat + 2 * nb_samples) / 3, 10)) + # Store the bars objects for enrichment plot + rects = [] + # For each sample/experiment + #for i in range(n_exp): + for sample_label in sample_labels: + # First barplot: percentage of reads in each categorie + n = sample_labels.index(sample_label) + #ax1.bar(index + i * bar_width, percentages[i], bar_width, + ax1.bar(index + n * bar_width, percentages[n], bar_width, + alpha=opacity, + #color=cmap[i], + color=cmap[n], + #label=sample_names[i], edgecolor="#FFFFFF", lw=0) + label=sample_label, edgecolor="#FFFFFF", lw=0) + # Second barplot: enrichment relative to the genome for each categ + # (the reads count in a categ is divided by the categ size in the genome) + rects.append(ax2.bar(index + n * bar_width, enrichment[n], bar_width, + alpha=opacity, + #color=cmap[i], + color=cmap[n], + #label=sample_names[i], edgecolor=cmap[i], lw=0)) + label=sample_label, edgecolor=cmap[n], lw=0)) + + ## Graphical options for the plot + # Adding of the legend + #if n_exp < 10: + if nb_samples < 10: + ax1.legend(loc="best", frameon=False) + legend_ncol = 1 + #elif n_exp < 19: + elif nb_samples < 19: + legend_ncol = 2 + else: + legend_ncol = 3 + ax1.legend(loc="best", frameon=False, ncol=legend_ncol) + ax2.legend(loc="best", frameon=False, ncol=legend_ncol) + # ax2.legend(loc='upper center',bbox_to_anchor=(0.5,-0.1), fancybox=True, shadow=True) + # Main titles + if title: + ax1.set_title(title) + else: + ax1.set_title(counts_type + " counts") + ax2.set_title(counts_type + " normalized counts") + + # Adding enrichment baseline + # ax2.axhline(y=0,color='black',linestyle='dashed',linewidth='1.5') + # Axes limits + ax1.set_xlim(-0.1, len(ordered_categs) + 0.1) + if len(sizes) == 1: ax1.set_xlim(-2, 3) + ax2.set_xlim(ax1.get_xlim()) + # Set axis limits (max/min values + 5% margin) + ax2_ymin = [] + ax2_ymax = [] + for sample_values in enrichment: + ax2_ymin.append(min(sample_values)) + ax2_ymax.append(max(sample_values)) + ax2_ymax = max(ax2_ymax) + ax2_ymin = min(ax2_ymin) + margin_top, margin_bottom = (abs(0.05 * ax2_ymax), abs(0.05 * ax2_ymin)) + ax1.set_ylim(0, ax1.get_ylim()[1] * 1.05) + if threshold: + threshold_bottom = -abs(float(threshold[0])) + 1 + threshold_top = float(threshold[1]) - 1 + + #for i in xrange(n_exp): + for n in xrange(nb_samples): + for y in xrange(n_cat): + #val = enrichment[i][y] + val = enrichment[n][y] + if not numpy.isnan(val) and not (threshold_bottom < val < threshold_top): + #rect = rects[i][y] + rect = rects[n][y] + rect_height = rect.get_height() + if rect.get_y() < 0: + diff = rect_height + threshold_bottom + rect.set_y(threshold_bottom) + ax2_ymin = threshold_bottom + margin_bottom = 0 + else: + diff = rect_height - threshold_top + ax2_ymax = threshold_top + margin_top = 0 + rect.set_height(rect.get_height() - diff) + if margin_top != 0 and margin_bottom != 0: + margin_top, margin_bottom = [max(margin_top, margin_bottom) for i in xrange(2)] + ax2.set_ylim(ax2_ymin - margin_bottom, ax2_ymax + margin_top) + # Y axis title + ax1.set_ylabel("Proportion of reads (%)") + ax2.set_ylabel("Enrichment relative to genome") + + + # Add the categories on the x-axis + #ax1.set_xticks(index + bar_width * n_exp / 2) + ax1.set_xticks(index + bar_width * nb_samples / 2) + #ax2.set_xticks(index + bar_width * n_exp / 2) + ax2.set_xticks(index + bar_width * nb_samples / 2) + if counts_type.lower() != "categories": + ax1.set_xticklabels(ordered_categs, rotation="30", ha="right") + ax2.set_xticklabels(ordered_categs, rotation="30", ha="right") + else: + ax1.set_xticklabels(xlabels) + ax2.set_xticklabels(xlabels) + + # Display fractions values in percentages + ax1.set_yticklabels([str(int(i * 100)) for i in ax1.get_yticks()]) + # Correct y-axis ticks labels for enrichment subplot + # ax2.set_yticklabels([str(i+1)+"$^{+1}$" if i>0 else 1 if i==0 else str(-(i-1))+"$^{-1}$" for i in ax2.get_yticks()]) + yticks = list(ax2.get_yticks()) + yticks = [yticks[i] - 1 if yticks[i] > 9 else yticks[i] + 1 if yticks[i] < -9 else yticks[i] for i in + xrange(len(yticks))] + ax2.set_yticks(yticks) + ax2.set_yticklabels([str(int(i + 1)) + "$^{+1}$" if i > 0 and i % 1 == 0 else str( + i + 1) + "$^{+1}$" if i > 0 else 1 if i == 0 else str( + int(-(i - 1))) + "$^{-1}$" if i < 0 and i % 1 == 0 else str(-(i - 1)) + "$^{-1}$" for i in ax2.get_yticks()]) + # ax2.set_yticklabels([i+1 if i>0 else 1 if i==0 else "$\\frac{1}{%s}$" %-(i-1) for i in ax2.get_yticks()]) + # Change appearance of 'antisense' bars on enrichment plot since we cannot calculate an enrichment for this artificial category + if "antisense_pos" in locals(): # ax2.text(antisense_pos+bar_width/2,ax2.get_ylim()[1]/10,'NA') + #for i in xrange(n_exp): + for n in xrange(nb_samples): + #rect = rects[i][antisense_pos] + rect = rects[n][antisense_pos] + rect.set_y(ax2.get_ylim()[0]) + rect.set_height(ax2.get_ylim()[1] - ax2.get_ylim()[0]) + rect.set_hatch("/") + rect.set_fill(False) + rect.set_linewidth(0) + # rect.set_color('lightgrey') + # rect.set_edgecolor('#EDEDED') + rect.set_color("#EDEDED") + #ax2.text(index[antisense_pos] + bar_width * n_exp / 2 - 0.1, (ax2_ymax + ax2_ymin) / 2, "NA") + ax2.text(index[antisense_pos] + bar_width * nb_samples / 2 - 0.1, (ax2_ymax + ax2_ymin) / 2, "NA") + # Add text for features absent in sample + #for i in xrange(n_exp): + for n in xrange(nb_samples): + for y in xrange(n_cat): + #if percentages[i][y] == 0: + if percentages[n][y] == 0: + txt = ax1.text(y + bar_width * (n + 0.5), 0.02, "Abs.", rotation="vertical", color=cmap[n], + horizontalalignment="center", verticalalignment="bottom") + txt.set_path_effects([PathEffects.Stroke(linewidth=0.5), PathEffects.Normal()]) + #elif enrichment[i][y] == 0: + elif enrichment[n][y] == 0: + #rects[i][y].set_linewidth(1) + rects[n][y].set_linewidth(1) + + # Remove top/right/bottom axes + for ax in [ax1, ax2]: + ax.spines["top"].set_visible(False) + ax.spines["right"].set_visible(False) + ax.spines["bottom"].set_visible(False) + ax.tick_params(axis="x", which="both", bottom="on", top="off", labelbottom="on") + ax.tick_params(axis="y", which="both", left="on", right="off", labelleft="on") + + + ### Add second axis with categ groups + annotate_group(categ_groups, label=None, ax=ax1) + annotate_group(categ_groups, label=None, ax=ax2) + + ### Adjust figure margins to + if counts_type.lower() == "categories": + plt.tight_layout(h_pad=5.0) + fig.subplots_adjust(bottom=0.1) + else: + plt.tight_layout() + + ## Showing the plot + if pdf: ## TODO: allow several output formats + pdf.savefig() + plt.close() + elif svg: + if svg == True: + plt.savefig(counts_type + ".svg") + else: + if os.path.splitext(svg)[1] == ".svg": + plt.savefig(".".join((os.path.splitext(svg)[0], counts_type, "svg"))) + else: + plt.savefig(".".join((svg, counts_type, "svg"))) + elif png: + if png == True: + plt.savefig(counts_type + ".png") + else: + if os.path.splitext(png)[1] == ".png": + plt.savefig(".".join((os.path.splitext(png)[0], counts_type, "png"))) + else: + plt.savefig(".".join((png, counts_type, "png"))) + else: + plt.show() + ## Save on disk it as a png image + # fig.savefig('image_output.png', dpi=300, format='png', bbox_extra_artists=(lgd,), bbox_inches='tight') + +def annotate_group(groups, ax=None, label=None, labeloffset=30): + """Annotates the categories with their parent group and add x-axis label""" + + def annotate(ax, name, left, right, y, pad): + """Draw the group annotation""" + arrow = ax.annotate(name, xy=(left, y), xycoords="data", + xytext=(right, y - pad), textcoords="data", + annotation_clip=False, verticalalignment="top", + horizontalalignment="center", linespacing=2.0, + arrowprops={'arrowstyle': "-", 'shrinkA': 0, 'shrinkB': 0, + 'connectionstyle': "angle,angleB=90,angleA=0,rad=5"} + ) + return arrow + + if ax is None: + ax = plt.gca() + level = 0 + for level in range(len(groups)): + grp = groups[level] + for name, coord in grp.items(): + ymin = ax.get_ylim()[0] - np.ptp(ax.get_ylim()) * 0.12 - np.ptp(ax.get_ylim()) * 0.05 * (level) + ypad = 0.01 * np.ptp(ax.get_ylim()) + xcenter = np.mean(coord) + annotate(ax, name, coord[0], xcenter, ymin, ypad) + annotate(ax, name, coord[1], xcenter, ymin, ypad) + + if label is not None: + # Define xlabel and position it according to the number of group levels + ax.annotate(label, + xy=(0.5, 0), xycoords="axes fraction", + xytext=(0, -labeloffset - (level + 1) * 15), textcoords="offset points", + verticalalignment="top", horizontalalignment="center") + + return + +def filter_categs_on_biotype(selected_biotype, cpt): + filtered_cpt = {} + for sample in cpt: + filtered_cpt[sample] = {} + for feature, count in cpt[sample].items(): + if feature[1] == selected_biotype: + filtered_cpt[sample][feature[0]] = count + return filtered_cpt + + +def unnecessary_param(param, message): + """ Function to display a warning on the standard error. """ + if param: + print >> sys.stderr, message + + +def usage_message(): + return """ + Generate genome indexes: + python ALFA.py -a GTF_FILE [-g GENOME_INDEX] + [--chr_len CHR_LENGTHS_FILE] + Process BAM file(s): + python ALFA.py -g GENOME_INDEX -i BAM1 LABEL1 [BAM2 LABEL2 ...] + [--bedgraph] [-s STRAND] + [-n] [--pdf output.pdf] + [-d {1,2,3,4}] [-t YMIN YMAX] + Index genome + process BAM: + python ALFA.py -a GTF_FILE [-g GENOME_INDEX] + -i BAM1 LABEL1 [BAM2 LABEL2 ...] + [--chr_len CHR_LENGTHS_FILE] + [--bedgraph][-s STRAND] + [-n] [--pdf output.pdf] + [-d {1,2,3,4}] [-t YMIN YMAX] + + Process previously created ALFA counts file(s): + python ALFA.py -c COUNTS1 [COUNTS2 ...] + [-s STRAND] + [-n] [--pdf output.pdf] + [-d {1,2,3,4}] [-t YMIN YMAX] + + """ + +########################################################################## +# MAIN # +########################################################################## + + +if __name__ == "__main__": + + #### Parse command line arguments and store them in the variable options + parser = argparse.ArgumentParser(formatter_class=argparse.RawTextHelpFormatter, usage=usage_message()) + parser.add_argument("--version", action="version", version="version 1.0", + help="Show ALFA version number and exit\n\n-----------\n\n") + # Options regarding the index + parser.add_argument("-g", "--genome_index", + help="Genome index files path and basename for existing index, or path and basename for new index creation\n\n") + parser.add_argument("-a", "--annotation", metavar="GTF_FILE", help="Genomic annotations file (GTF format)\n\n") + parser.add_argument("--chr_len", help="Tabulated file containing chromosome names and lengths\n\n-----------\n\n") + + # Options regarding the intersection step + #parser.add_argument('-i', '--input', '--bam', dest='input', metavar=('BAM_FILE1 LABEL1', ""), nargs='+', + #help='Input BAM file(s) and label(s). The BAM files must be sorted by position.\n\n') + parser.add_argument("--bam", metavar=("BAM_FILE1 LABEL1", ""), nargs="+", + help="Input BAM file(s) and label(s). The BAM files must be sorted by position.\n\n") ## MB: position AND chr?? + # parser.add_argument('--bedgraph', action='store_const',default = False, const = True, help="Use this options if your input file(s) is(are) already in bedgraph format\n\n") + #parser.add_argument('--bedgraph', dest='bedgraph', action='store_const', default=False, const=True, + #help="Use this options if your input file(s) is(are) already in bedgraph format\n\n") + parser.add_argument("--bedgraph", metavar=("BEDGRAPH_FILE1 LABEL1", ""), nargs="+", + help="Use this options if your input is/are BedGraph file(s).\n\n") + parser.add_argument("-c", "--counts", metavar=("COUNTS_FILE", ""), nargs="+", + help="Use this options instead of '-i/--input' to provide ALFA counts files as input \ninstead of bam/bedgraph files.\n\n") + #parser.add_argument('-s', '--strandness', dest="strandness", nargs=1, action='store', default=['unstranded'], choices=['unstranded', 'forward', 'reverse', 'fr-firststrand', 'fr-secondstrand'], metavar="", help="Library orientation. Choose within: 'unstranded', 'forward'/'fr-firststrand' \nor 'reverse'/'fr-secondstrand'. (Default: 'unstranded')\n\n-----------\n\n") + parser.add_argument("-s", "--strandness", default="unstranded", + choices=["unstranded", "forward", "reverse", "fr-firststrand", "fr-secondstrand"], metavar="", + help="Library orientation. Choose within: 'unstranded', " + "'forward'/'fr-firststrand' \nor 'reverse'/'fr-secondstrand'. " + "(Default: 'unstranded')\n\n-----------\n\n") + + # Options regarding the plot + parser.add_argument("--biotype_filter", help=argparse.SUPPRESS) # "Make an extra plot of categories distribution using only counts of the specified biotype." ## MB: TO DISPLAY (no suppress) + parser.add_argument("-d", "--categories_depth", type=int, default=3, choices=range(1, 5), + help="Use this option to set the hierarchical level that will be considered in the GTF file (default=3): \n(1) gene,intergenic; \n(2) intron,exon,intergenic; \n(3) 5'UTR,CDS,3'UTR,intron,intergenic; \n(4) start_codon,5'UTR,CDS,3'UTR,stop_codon,intron,intergenic. \n\n") + parser.add_argument("--pdf", nargs="?", const="ALFA_plots.pdf", + help="Save produced plots in PDF format at specified path ('categories_plots.pdf' if no argument provided).\n\n") + parser.add_argument("--png", nargs="?", const="ALFA_plots.png", + help="Save produced plots in PNG format with provided argument as basename \nor 'categories.png' and 'biotypes.png' if no argument provided.\n\n") + parser.add_argument("--svg", nargs="?", const="ALFA_plots.svg", + help="Save produced plots in SVG format with provided argument as basename \nor 'categories.svg' and 'biotypes.svg' if no argument provided.\n\n") + parser.add_argument("-n", "--no_display", action="store_const", const=True, default=False, help="Do not display plots.\n\n") # We have to add "const=None" to avoid a bug in argparse + parser.add_argument("-t", "--threshold", dest="threshold", nargs=2, metavar=("ymin", "ymax"), type=float, + help="Set axis limits for enrichment plots.\n\n") + + if len(sys.argv) == 1: + parser.print_usage() + sys.exit(1) + + options = parser.parse_args() + + ''' + # Booleans for steps to be executed + make_index = False + intersect_reads = False + process_counts = False + + #### Check arguments conformity and define which steps have to be performed + print "### Checking parameters" + if options.counts: + # Aucun autre argument requis, precise that the other won't be used (if this is true!!) + # Vérifier extension input + + # Action: Only do the plot + process_counts = True + else: + if options.annotation: + # If '-gi' parameter is present + if options.genome_index: + genome_index_basename = options.genome_index + else: + # Otherwise the GTF filename without extension will be the basename + genome_index_basename = options.annotation.split("/")[-1].split(".gtf")[0] + # Check if the indexes were already created and warn the user + if os.path.isfile(genome_index_basename + ".stranded.index"): + if options.input: + print >> sys.stderr, "Warning: an index file named '%s' already exists and will be used. If you want to create a new index, please delete this file or specify an other path." % ( + genome_index_basename + ".stranded.index") + else: + sys.exit( + "Error: an index file named %s already exists. If you want to create a new index, please delete this file or specify an other path.\n" % ( + genome_index_basename + ".stranded.index")) + # Create them otherwise + else: + make_index = True + # If the index is already done + if options.input: + # Required arguments are the input and the genome_index + if 'genome_index_basename' not in locals(): + required_arg(options.genome_index, "-g/--genome_index") + genome_index_basename = options.genome_index + required_arg(options.input, "-i/--input/--bam") + for i in xrange(0, len(options.input), 2): + # Check whether the input file exists + try: + open(options.input[i]) + except IOError: + sys.exit("Error: the input file " + options.input[i] + " was not found. Aborting.") + # Check whether the input file extensions are 'bam', 'bedgraph' or 'bg' and the label extension are no + try: + extension = os.path.splitext(options.input[i + 1])[1] + if extension in ('.bam', '.bedgraph', '.bg'): + sys.exit("Error: it seems input files and associated labels are not correctly provided.\n\ + Make sure to follow the expected format : -i Input_file1 Label1 [Input_file2 Label2 ...].") + except: + sys.exit( + "Error: it seems input files and associated labels are not correctly provided.\nMake sure to follow the expected format : -i Input_file1 Label1 [Input_file2 Label2 ...].") + + intersect_reads = True + # Vérifier input's extension + # TODO + if not (options.counts or options.input or options.annotation): + sys.exit( + "\nError : some arguments are missing At least '-a', '-c' or '-i' is required. Please refer to help (-h/--help) and usage cases for more details.\n") + if not options.counts: + # Declare genome_index variables + stranded_genome_index = genome_index_basename + ".stranded.index" + unstranded_genome_index = genome_index_basename + ".unstranded.index" + if options.strandness[0] == "unstranded": + genome_index = unstranded_genome_index + else: + genome_index = stranded_genome_index + ''' + #### Initialization of some variables + + # Miscellaneous variables + reverse_strand = {"+": "-", "-": "+"} + samples = collections.OrderedDict() # Structure: {<label>: [<filename1>(, <filename2>)]} # Example: {'Toy': ['toy.bam']} + + #Sample labels and file paths + labels = [] + bams = [] + bedgraphs = [] + count_files = [] + + + # Initializing the category priority order, coding biotypes and the final list + # prios = {"start_codon": 7, "stop_codon": 7, "five_prime_utr": 6, "three_prime_utr": 6, "UTR": 6, "CDS": 5, "exon": 4, + # "transcript": 3, "gene": 2, "antisense": 1, "intergenic": 0} + prios = {"start_codon": 4, "stop_codon": 4, "five_prime_utr": 3, "three_prime_utr": 3, "UTR": 3, "CDS": 3, + "exon": 2, "intron": 2, "transcript": 1, "gene": 1, "antisense": 0, "intergenic": -1} + + biotype_prios = None + # biotype_prios = {"protein_coding":1, "miRNA":2} + + # Possibles groups of categories to plot + # categs_group1 = {"start": ["start_codon"], "5UTR": ["five_prime_utr", "UTR"], "CDS": ["CDS", "exon"], + # "3UTR": ["three_prime_utr"], "stop": ["stop_codon"], "introns": ["transcript", "gene"], + # "intergenic": ["intergenic"], "antisense": ["antisense"]} + # categs_group2 = {"5UTR": ["five_prime_utr", "UTR"], "CDS": ["CDS", "exon", "start_codon", "stop_codon"], + # "3UTR": ["three_prime_utr"], "introns": ["transcript", "gene"], "intergenic": ["intergenic"], + # "antisense": ["antisense"]} + # categs_group3 = {"exons": ["five_prime_utr", "three_prime_utr", "UTR", "CDS", "exon", "start_codon", "stop_codon"], + # "introns": ["transcript", "gene"], "intergenic": ["intergenic"], "antisense": ["antisense"]} + # categs_group4 = { + # "gene": ["five_prime_utr", "three_prime_utr", "UTR", "CDS", "exon", "start_codon", "stop_codon", "transcript", + # "gene"], "intergenic": ["intergenic"], "antisense": ["antisense"]} + + + categs_level1 = {"gene": ["five_prime_utr", "three_prime_utr", "UTR", "CDS", "exon", "intron", "start_codon", + "stop_codon", "transcript", "gene"], + "intergenic": ["intergenic"], + "antisense": ["antisense"]} + + categs_level2 = {"exons": ["five_prime_utr", "three_prime_utr", "UTR", "CDS", "exon", "start_codon", "stop_codon"], + "introns": ["intron"], + "undescribed_genes": ["transcript", "gene"], + "intergenic": ["intergenic"], + "antisense": ["antisense"]} + + categs_level3 = {"5UTR": ["five_prime_utr", "UTR"], + "CDS": ["CDS", "start_codon", "stop_codon"], + "3UTR": ["three_prime_utr"], + "undescribed_exons": ["exon"], + "introns": ["intron"], + "undescribed_genes": ["transcript", "gene"], + "intergenic": ["intergenic"], + "antisense": ["antisense"]} + + categs_level4 = {"5UTR": ["five_prime_utr", "UTR"], + "start": ["start_codon"], + "stop": ["stop_codon"], + "CDS_body": ["CDS"], + "undescribed_CDS": [], #TODO: implement CDS/undescribed_CDS distinction + "3UTR": ["three_prime_utr"], + "undescribed_exons": ["exon"], + "introns": ["intron"], + "undescribed_genes": ["transcript", "gene"], + "intergenic": ["intergenic"], + "antisense": ["antisense"]} + + # categs_groups = [categs_group4, categs_group3, categs_group2, categs_group1] # Order and merging for the final plot + categs_levels = [categs_level1, categs_level2, categs_level3, categs_level4] + + parent_categ_level1 = [] + parent_categ_level2 = [{"gene":[0.5,2.5]}] + parent_categ_level3 = [{"exon":[0.5,3.5]}, {"gene":[0.5,5.5]}] + parent_categ_level4 = [{"CDS":[1.5,4.5]}, {"exon":[0.5,6.5]},{"gene":[0.5,8.5]}] + parent_categ_groups = [parent_categ_level1, parent_categ_level2, parent_categ_level3, parent_categ_level4] + + cat_list = ["5UTR", "start", "CDS", "CDS_body", "stop", "undescribed_CDS", "3UTR", "exons", "undescribed_exons", "introns", "gene", "undescribed_genes", "intergenic", "antisense"] + + # biotypes list + biotypes = {"protein_coding", "polymorphic_pseudogene", "TR_C_gene", "TR_D_gene", "TR_J_gene", "TR_V_gene", "IG_C_gene", + "IG_D_gene", "IG_J_gene", "IG_V_gene", "3prime_overlapping_ncrna", "lincRNA", "macro_lncRNA", "miRNA", + "misc_RNA", "Mt_rRNA", "Mt_tRNA", "processed_transcript", "ribozyme", "rRNA", "scaRNA", "sense_intronic", + "sense_overlapping", "snoRNA", "snRNA", "sRNA", "TEC", "vaultRNA", "antisense", + "transcribed_processed_pseudogene", "transcribed_unitary_pseudogene", "transcribed_unprocessed_pseudogene", + "translated_unprocessed_pseudogene", "TR_J_pseudogene", "TR_V_pseudogene", "unitary_pseudogene", + "unprocessed_pseudogene", "processed_pseudogene", "IG_C_pseudogene", "IG_J_pseudogene", "IG_V_pseudogene", + "pseudogene", "ncRNA", "tRNA"} # Type: set (to access quickly) + + # Grouping of biotypes: + biotypes_group1 = {"protein_coding": ["protein_coding"], + "pseudogenes": ["polymorphic_pseudogene", "transcribed_processed_pseudogene", + "transcribed_unitary_pseudogene", "transcribed_unprocessed_pseudogene", + "translated_unprocessed_pseudogene", "TR_J_pseudogene", "TR_V_pseudogene", + "unitary_pseudogene", "unprocessed_pseudogene", "processed_pseudogene", + "IG_C_pseudogene", "IG_J_pseudogene", "IG_V_pseudogene", "pseudogene"], + "TR": ["TR_C_gene", "TR_D_gene", "TR_J_gene", "TR_V_gene"], + "IG": ["IG_C_gene", "IG_D_gene", "IG_J_gene", "IG_V_gene"], \ + "MT_RNA": ["Mt_rRNA", "Mt_tRNA"], \ + "ncRNA": ["lincRNA", "macro_lncRNA", "3prime_overlapping_ncrna", "ncRNA"], \ + "others": ["misc_RNA", "processed_transcript", "ribozyme", "scaRNA", "sense_intronic", + "sense_overlapping", "TEC", "vaultRNA"], + "antisense": ["antisense"]} + for biot in ["miRNA", "snoRNA", "snRNA", "rRNA", "sRNA", "tRNA"]: + biotypes_group1[biot] = [biot] + + # Initializing the unknown features list + unknown_cat = set() + unknown_biot= set() + + # Initializing the genome category counter dict + cpt_genome = {} + ''' + if process_counts: + #### If input files are the categories counts, just load them and continue to recategorization step + cpt, cpt_genome, samples_names = read_counts_files(options.counts) + else: + #### Create genome index if needed and get the sizes of categories + index_chrom_list = [] + if make_index: + #### Get the chromosome lengths + lengths = get_chromosome_lengths(options) + # Generating the genome index files if the user didn't provide them + create_genome_index(options.annotation, unstranded_genome_index, stranded_genome_index, cpt_genome, prios, + biotypes, lengths) + else: + # Retrieving chromosome names saved in index + index_chrom_list = get_chromosome_names_in_index(genome_index) + + + # print '\nChr lengths:', lengths + + if intersect_reads: + # If the indexes already exist, read them to compute the sizes of the categories in the genome and retrieve the chromosome lengths + if not make_index: + print "\n### Reading genome indexes\n", + sys.stdout.flush() + lengths = {} + with open(genome_index, 'r') as genome_index_file: + for line in genome_index_file: + if line[0] == "#": + lengths[line.split('\t')[0][1:]] = int(line.split('\t')[1]) + else: + add_info(cpt_genome, line.rstrip().split('\t')[4:], line.split('\t')[1], line.split('\t')[2], + biotype_prios=None, categ_prios=prios) + + #print 'Indexed chromosomes: ' + ', '.join((sorted(index_chrom_list))) + index_chrom_list.sort(key=alphanum_key) + print 'Indexed chromosomes: ' + ', '.join((index_chrom_list)) + + #### Computing the genome intergenic count: sum of the chr lengths minus sum of the genome annotated intervals + cpt_genome[('intergenic', 'intergenic')] = sum(lengths.itervalues()) - sum( + [v for x, v in cpt_genome.iteritems() if x != ('antisense', 'antisense')]) + if not make_index: + print "Done!" + # print '\nGenome category counts:' + # for key,val in cpt_genome.iteritems(): + # print key,"\t",val + + + #### Create the Bedgraph files if needed and get the files list + + if not options.bedgraph: + # Generating the BEDGRAPH files is the user provided BAM file(s) and get the samples labels (this names will be used in the plot legend) + samples_files, samples_names = create_bedgraph_files(options.input, options.strandness[0]) + else: + # Just initialize the files list with the bedgraph paths + # samples_files = [options.input[i] for i in range(0,len(options.input),2)] + samples_files = [re.sub('.bedgraph$', '', options.input[i]) for i in range(0, len(options.input), 2)] + # and get the labels + samples_names = [options.input[i] for i in range(1, len(options.input), 2)] + #### Retrieving chromosome names saved in index + #chrom_list = get_chromosome_names_in_index(genome_index) + #### Processing the BEDGRAPH files: intersecting the bedgraph with the genome index and count the number of aligned positions in each category + cpt = intersect_bedgraphs_and_index_to_counts_categories(samples_files, samples_names, prios, genome_index, + options.strandness[0], biotype_prios=None) + + #### Write the counts on disk + write_counts_in_files(cpt, cpt_genome) + + if not (intersect_reads or process_counts) or (options.quiet and options.pdf == False): + quit("\n### End of program") + print "\n### Generating plots" + # Updating the biotypes lists (biotypes and 'biotype_group1'): adding the 'unknow biotypes' found in gtf/index + if unknown_feature == []: # 'unknown_feature' is define only during the index generation + # Browse the feature to determine whether some biotypes are 'unknown' + for sample, counts in cpt.items(): + for (cat, biot) in counts: + if biot not in biotypes and cat not in unknown_feature: + unknown_feature.append(biot) + for new_biot in unknown_feature: + biotypes.add(new_biot) + biotypes_group1["others"].append(new_biot) + biotypes = sorted(biotypes) + # move antisense categ to the end of the list + biotypes.remove('antisense') + biotypes.append('antisense') + biotypes_group1 = sorted(biotypes_group1) + + # print '\nCounts for every category/biotype pair: ',cpt + + # Generating plots + if options.pdf != False: + if options.pdf == None: + options.pdf = "categories_plots.pdf" + pdf = PdfPages(options.pdf) + else: + pdf = False + + selected_biotype = None + if options.biotype_filter: + options.biotype_filter = options.biotype_filter[0] + for sample in cpt: + for feature in cpt[sample]: + biotype = feature[1] + if options.biotype_filter.lower() == biotype.lower(): + selected_biotype = biotype + break + if selected_biotype == None: + print "\nError: biotype '" + options.biotype_filter + "' not found. Please check the biotype name and that this biotype exists in your sample(s)." + sys.exit() + + # Print a warning message if the UTRs are not specified as 5' or 3' (they will be ploted as 5'UTR) + if 'UTR' in [categ[0] for counts in cpt.values() for categ in counts.keys()]: + print "\nWARNING: (some) 5'UTR/3'UTR are not precisely defined. Consequently, positions annotated as "UTR" will be counted as "5'UTR"\n" +##MB + #### Make the plot by categories + #### Recategorizing with the final categories + final_cats = categs_groups[options.categories_depth - 1] + final_cat_cpt, final_genome_cpt, filtered_cat_cpt = group_counts_by_categ(cpt, cpt_genome, final_cats, selected_biotype) + #### Display the distribution of specified categories (or biotypes) in samples on a barplot + # Remove the 'antisense' category if the library type is 'unstranded' + for dic in cpt.values(): + if ('antisense', 'antisense') in dic.keys(): break + else: + cat_list.remove('antisense') + make_plot(cat_list, samples_names, final_cat_cpt, final_genome_cpt, pdf, "categories", options.threshold, + svg=options.svg, png=options.png) + if selected_biotype: + make_plot(cat_list, samples_names, filtered_cat_cpt, final_genome_cpt, pdf, "categories", options.threshold, + title="Categories distribution for '" + selected_biotype + "' biotype", svg=options.svg, png=options.png) + + #### Make the plot by biotypes + #### Recategorizing with the final categories + final_cat_cpt, final_genome_cpt = group_counts_by_biotype(cpt, cpt_genome, biotypes) + #### Display the distribution of specified categories (or biotypes) in samples on a barplot + make_plot(biotypes, samples_names, final_cat_cpt, final_genome_cpt, pdf, "biotypes", options.threshold, svg=options.svg, + png=options.png) + + ##### Recategorizing with the final categories + # final_cat_cpt,final_genome_cpt = group_counts_by_biotype(cpt,cpt_genome,biotypes_group1) + ##### Display the distribution of specified categories (or biotypes) in samples on a barplot + # make_plot(biotypes_group1,samples_names,final_cat_cpt,final_genome_cpt,pdf,"Biotype groups", options.threshold, title="Biotypes distribution in mapped reads \n(biotypes are grouped by 'family')", svg = options.svg, png = options.png) + + + if options.pdf: + pdf.close() + print "\n### Plots saved in pdf file: %s" % options.pdf + + print "\n### End of program" + ''' + print "### ALFA ###" + if not (options.annotation or options.bam or options.bedgraph or options.counts): + print >> sys.stderr, "\nError: At least one argument among '-a/--annotation', '--bam', '--bedgraph' or " \ + "'-c/--counts' is required. Please refer to help (-h/--help) and usage cases for more " \ + "details.\n" + parser.print_usage() + sys.exit() + + ## Getting the steps to execute and checking parameters + + # Getting the steps to execute + generate_indexes = False + generate_BedGraph = False + intersect_indexes_BedGraph = False + generate_plot = False + + # Checking parameters for the step: indexes generation + if options.annotation: + generate_indexes = True + elif options.chr_len: + unnecessary_param(options.chr_len, "Warning: the parameter '--chr_len' will not be used because the indexes generation step will not be performed.") + + # Checking parameters for the step: BedGraph files generation + if options.bam: + if options.bedgraph: + sys.exit("\nError: parameters '--bam' and '--bedgraph' provided, only one should be.\n### End of program") + else: + if not generate_indexes and not options.genome_index: + sys.exit("\nError: parameter '-g/--genome_index' should be provided.\n### End of program") + # Checking the input BAM files + for i in xrange(0, len(options.bam), 2): + # Check whether the BAM file exists + try: + open(options.bam[i]) + except IOError: + sys.exit("\nError: the BAM file " + options.bam[i] + " was not found.\n### End of program") + except IndexError: + sys.exit("\nError: BAM files and associated labels are not correctly provided.\n" + "Make sure to follow the expected format: --bam BAM_file1 Label1 [BAM_file2 Label2 ...]." + "\n### End of program ###") + + # Check whether the BAM file extension in 'bam' + if not options.bam[i].endswith(".bam"): + sys.exit("\nError: at least one BAM file hasn't a '.bam' extension.\n### End of program ###") + # Check whether the labels hasn't a "bam" extension + try: + if options.bam[i + 1].endswith(".bam"): + sys.exit("\nError: at least one label for a BAM file has a '.bam' extension.\n" + "Make sure to follow the expected format: --bam BAM_file1 Label1 [BAM_file2 Label2 ...]." + "\n### End of program ###") + except IndexError: + sys.exit("\nError: BAM files and associated labels are not correctly provided.\n" + "Make sure to follow the expected format: --bam BAM_file1 Label1 [BAM_file2 Label2 ...]." + "\n### End of program ###") + # Get label and replace invalid characters by "_" + label = "_".join(re.findall(r"[\w\-']+", options.bam[i + 1])) + # Check whether the BedGraph file(s) and counts file that will be generated already exists + if options.strandness == "unstranded": + existing_file(label + ".bedgraph") + existing_file(label + ".unstranded.feature_counts.tsv") + else: + existing_file(label + ".plus.bedgraph") + existing_file(label + ".minus.bedgraph") + existing_file(label + ".stranded.feature_counts.tsv") + # Register the sample label and filename + labels.append(label) + bams.append(options.bam[i]) + # Set this step + the intersection one as tasks to process + generate_BedGraph = True + intersect_indexes_BedGraph = True + + # Checking parameters for the step: indexes and BedGraph files intersection + if options.bedgraph: + if not generate_indexes and not options.genome_index: + sys.exit("\nError: parameter '-g/--genome_index' should be provided.\n### End of program") + if options.strandness == "unstranded": + sample_file_nb = 2 + else: + sample_file_nb = 3 + # Checking the input BedGraph files + for i in xrange(0, len(options.bedgraph), sample_file_nb): + # Check whether the BedGraph file(s) exists + for j in xrange(0,sample_file_nb - 1): + try: + open(options.bedgraph[i + j]) + except IOError: + if not (options.bedgraph[i + j].endswith(".bedgraph") or options.bedgraph[i + j].endswith(".bg")): + sys.exit("\nError: it looks like BedGraph files and associated labels are not correctly provided.\n" + "Make sure to follow the expected format: --bedgraph BedGraph_file1 Label1 [BedGraph_file2 Label2 ...]." + "\n### End of program ###") + else: + sys.exit("\nError: the BedGraph file " + options.bedgraph[i + j] + " was not found.\n### End of program") + except IndexError: + sys.exit("\nError: it looks like BedGraph files and associated labels are not correctly provided.\n" + "Make sure to follow the expected format: --bedgraph BedGraph_file1 Label1 [BedGraph_file2 Label2 ...]." + "\n### End of program ###") + # Check whether the BedGraph file extension is "bedgraph"/"bg" + if not (options.bedgraph[i + j].endswith(".bedgraph") or options.bedgraph[i + j].endswith(".bg")): + sys.exit("\nError: the BedGrapg file" + options.bedgraph[i + j] + " hasn't a '.bedgraph'/'.bg' extension." + "\n### End of program ###") + # Check whether the labels hasn't a "bedgraph"/"bg" extension + try: + if options.bedgraph[i + sample_file_nb - 1].endswith('.bedgraph') or options.bedgraph[i + sample_file_nb - 1].endswith('.bg'): + sys.exit("\nError: the label " + options.bedgraph[i + sample_file_nb - 1] + " has a '.bedgraph'/'.bg' extension.\n" + "Make sure to follow the expected format: " + "--bedgraph BedGraph_file1 Label1 [BedGraph_file2 Label2 ...]." + "\n### End of program ###") + except IndexError: + sys.exit("\nError: it looks like BedGraph files and associated labels are not correctly provided.\n" + "Make sure to follow the expected format: --bedgraph BedGraph_file1 Label1 [BedGraph_file2 Label2 ...]." + "\n### End of program ###") + # Register the sample label and filename(s) + bedgraphs.append(re.sub("(.(plus|minus))?.((bg)|(bedgraph))", "", options.bedgraph[i])) + label = "_".join(re.findall(r"[\w\-']+", options.bedgraph[i + sample_file_nb - 1])) + labels.append(label) + # Check whether the count file(s) that will be created already exists + if options.strandness == "unstranded": + existing_file(label + ".unstranded.feature_counts.tsv") + else: + existing_file(label + ".stranded.feature_counts.tsv") + # Set this step as a task to process + intersect_indexes_BedGraph = True + + # Checking parameters for the step: plot generation + if options.counts: + # Checking unnecessary parameters + unnecessary_param(options.annotation, "Warning: the parameter '-a/--annotation' will not be used because the counts are already provided.") + generate_indexes = False + unnecessary_param(options.genome_index, "Warning: the parameter '-g/--genome_index' will not be used because the counts are already provided.") + unnecessary_param(options.bam, "Warning: the parameter '--bam' will not be used because the counts are already provided.") + generate_BedGraph = False + unnecessary_param(options.bedgraph, "Warning: the parameter '--bedgraph' will not be used because the counts are already provided.") + intersect_indexes_BedGraph = False + # Registering the sample labels and filenames + for sample in options.counts: + label = os.path.basename(sample) + label = re.sub('(.(un)?stranded)?.feature_counts.tsv', '', label) + label = "_".join(re.findall(r"[\w\-']+", label)) + count_files.append(sample) + labels.append(label) + else: + # Generating the genome index filenames + index_chrom_list = [] # Not a set because we need to sort it according to the chromosome names later + lengths = {} + if options.genome_index: + genome_index_basename = options.genome_index + elif options.annotation: + # Otherwise the GTF filename without extension will be the basename + genome_index_basename = options.annotation.split("/")[-1].split(".gtf")[0] + stranded_genome_index = genome_index_basename + ".stranded.index" + unstranded_genome_index = genome_index_basename + ".unstranded.index" + if options.strandness == "unstranded": + genome_index = unstranded_genome_index + else: + genome_index = stranded_genome_index + # If no plot is displayed or saved, plot parameters are useless + if (options.no_display and not (options.pdf or options.png or options.svg)) or not(intersect_indexes_BedGraph or options.counts): + # unnecessary_param(options.categories_depth, "Warning: the parameter '-d/--categories_depth' will not be used because no plots will be displayed or saved.") + # # Cannot be tested because options.categories_depth has always a value (default value if option not specified by user) + unnecessary_param(options.threshold, "Warning: the parameter '-t/--threshold' will not be used because no plots will be displayed or saved.") + unnecessary_param(options.biotype_filter, "Warning: the parameter '--biotype_filter' will not be used because no plots will be displayed or saved.") + if options.counts: + sys.exit("Error: there is nothing to do (counts are provided and no display or plot saving is required") + else: + ## [BN] X server check + # if not X server: + #options.no_display = True + #Message + try: + x_server = os.environ['DISPLAY'] + generate_plot = True + except: + x_server = False + if options.counts: + sys.exit("Error: your current configuration does not allow graphical interface ('DISPLAY' variable is not set on your system).\nExiting") + else: + print >> sys.stderr, "WARNING: your current configuration does not allow graphical interface ('DISPLAY' variable is not set on your system).\nPlotting step will not be performed." + + ## Executing the step(s) + + # Indexes generation + if generate_indexes: + # Checking if the index files already exist + if os.path.isfile(stranded_genome_index): + sys.exit("\nError: index files named '" + genome_index_basename + + ".stranded.index' already exists but you provided a GTF file. If you want to generate a new index, " + "please delete these files or specify an other path.\n### End of program") + # Running the index generation commands + print "# Generating the genome index files" + # Getting chromosomes lengths + lengths = get_chromosome_lengths() + # Generating the index files + generate_genome_index(options.annotation, unstranded_genome_index, stranded_genome_index, lengths) + # Displaying the list of indexed chromosomes + index_chrom_list.sort(key=alphanum_key) + print "Indexed chromosomes: " + ", ".join(index_chrom_list) + if generate_indexes or not options.counts: + # Getting index info + read_index() + # Computing the genome intergenic count: sum of the chr lengths minus sum of the genome annotated intervals + cpt_genome[("intergenic", "intergenic")] = sum(lengths.values()) - sum([v for x, v in cpt_genome.iteritems() if x != ("antisense", "antisense")]) + + # BedGraph files generation + if generate_BedGraph: + print "# Generating the BedGraph files" + #sample_files, sample_labels = generate_bedgraph_files() + generate_bedgraph_files(labels, bams) + + # Indexes and BedGraph files intersection + if intersect_indexes_BedGraph: + print "# Intersecting index and BedGraph files" + cpt = intersect_bedgraphs_and_index_to_counts_categories(labels, bedgraphs) + # Write the counts to an output file + write_counts_in_files(cpt, cpt_genome) + + ## Plot generation ## MB: all the section still to review + if generate_plot: + print "# Generating plots" + # If input files are the categories counts, the first step is to load them + if options.counts: + #cpt, cpt_genome, sample_names = read_counts(options.counts) + cpt, cpt_genome = read_counts(labels, count_files) + # Managing the unknown biotypes + for sample_label, counters in cpt.items(): + for (cat, biot) in counters: + if biot not in biotypes: + unknown_biot.add(biot) + for biot in unknown_biot: + biotypes.add(biot) + biotypes_group1["others"].append(biot) + biotypes = sorted(biotypes) + # Moving antisense cat to the end of the list + biotypes.remove("antisense") + biotypes.append("antisense") + biotypes_group1 = sorted(biotypes_group1) + # Filtering biotypes if necessary + filtered_biotype = None + if options.biotype_filter: + for sample_label in cpt: + for feature in cpt[sample_label]: + biotype = feature[1] + if options.biotype_filter.lower() == biotype.lower(): + selected_biotype = biotype + break + if filtered_biotype: + print "\nWarning: biotype '" + options.biotype_filter + "' not found. Please check the biotype name and that this biotype exists in your sample(s)." + # Setting the plots filenames + if options.pdf: ## MB: Do the same for svg and png?? + pdf = PdfPages(options.pdf) + else: + pdf = False + ## Generate the categories plot + # Recategorizing within the final categories and plot generation + final_cats = categs_levels[options.categories_depth - 1] + parent_categs = parent_categ_groups[options.categories_depth - 1] + final_cat_cpt, final_genome_cpt, filtered_cat_cpt = group_counts_by_categ(cpt, cpt_genome, final_cats, filtered_biotype) + # Remove the "antisense" category if the library type is "unstranded" ## MB: if options.strandness == "unstranded": cat_list.remove("antisense")?? + for dic in cpt.values(): + if ("antisense", "antisense") in dic.keys(): break + else: + cat_list.remove("antisense") + #make_plot(labels, cat_list, sample_labels, final_cat_cpt, final_genome_cpt, pdf, "categories", options.threshold, svg=options.svg, png=options.png) + make_plot(labels, cat_list, final_cat_cpt, final_genome_cpt, pdf, "Categories", options.threshold, svg=options.svg, png=options.png, categ_groups= parent_categs) + if filtered_biotype: + #make_plot(labels, cat_list, sample_labels, filtered_cat_cpt, final_genome_cpt, pdf, "categories", options.threshold, title="Categories distribution for '" + filtered_biotype + "' biotype", svg=options.svg, png=options.png) + make_plot(labels, cat_list, filtered_cat_cpt, final_genome_cpt, pdf, "Categories", options.threshold, title="Categories distribution for '" + filtered_biotype + "' biotype", svg=options.svg, png=options.png, categ_groups= parent_categs) + ## Generate the biotypes plot + # Recategorization within the final biotypes and plot generation + final_cat_cpt, final_genome_cpt = group_counts_by_biotype(cpt, cpt_genome, biotypes) + #make_plot(biotypes, sample_labels, final_cat_cpt, final_genome_cpt, pdf, "biotypes", options.threshold, svg=options.svg, png=options.png) + make_plot(labels, biotypes, final_cat_cpt, final_genome_cpt, pdf, "Biotypes", options.threshold, svg=options.svg, png=options.png) + + + print "### End of program ###" \ No newline at end of file
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/ALFA.xml Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,331 @@ +<tool id="alfa" name="ALFA" version="0.1.0"> + <description>- Plot the distribution of the genomic features captured by aligned reads </description> + + <!-- ALFA requires bedtools suite v2.20.0 and above --> + <requirements> + <requirement type="package" version="2.24">bedtools</requirement> + <requirement type="package" version="1.2">samtools</requirement> + <requirement type="package" version="1.4">matplotlib</requirement> + </requirements> + + <command interpreter="python"> + <![CDATA[ + ALFA_wrapper.py + + --project_name "${projectName}" + + ##__INPUT 1: ANNOTATION OF THE SEQ/GENOME__## + #if str ( $annotation.annotationSource['annotationSourceSelection'] ) == "index" + --index "$annotation.annotationSource['strandedIndex']" "$annotation.annotationSource['unstrandedIndex']" + #else if str ( $annotation.annotationSource['annotationSourceSelection'] ) == "built_in_index" + --bi_index "$annotation.annotationSource.built_in_index_prefix.fields.prefix" + #else + --annotation "$annotation.annotationSource['annotationFile']" + #end if + + ##__INPUT 2: ALIGNED READS__## + --reads_format $reads.readsType['readsTypeSelection'] + --reads + #for $i, $r in enumerate ( $reads.readsType['readsList'] ) + "__fname__$r.readsFile" + "__label__$r.readsLabel" + #end for + --strandness $reads['strandness'] + + ##__OUTPUT FILES__## + #if str ( $outputFiles['plot'] ) == "True" + #if str ( $outputOptions['plotFormat'] ) == "pdf" + --output_pdf "$outputPdf" + #else if str ( $outputOptions['plotFormat'] ) == "png" + --output_png "$outputCategoriesPng" "$outputBiotypesPng" + #else + --output_svg "$outputCategoriesSvg" "$outputBiotypesSvg" + #end if + #end if + #if str ( $outputFiles['countFile'] ) == "True" + --output_count "$outputCountFile" + #end if + #if str ( $outputFiles['index'] ) == "True" + --output_index "$outputStrandedIndex" "$outputUnstrandedIndex" + #end if + + ##__OUTPUT OPTIONS__## + --categories_depth $outputOptions['categoriesDepth'] + #if str ( $outputFiles['plot'] ) == "True" + --plot_format $outputOptions['plotFormat'] + #if str ( $outputOptions.plotThreshold['plotThresholdChoice'] ) == "True" + --threshold $outputOptions.plotThreshold.yMin $outputOptions.plotThreshold.yMax + #end if + #end if + + --log_report "$logReport" + --tool_dir "$__tool_directory__" + ]]> + </command> + <inputs> + <param name="projectName" value="ALFA" type="text" size="20" label="Project Name"> + <validator type="empty_field" message="Please, specify a name for your project."/> + </param> + + <section name="annotation" title="INPUT 1: Annotation of your genome / sequence" expanded="True"> + <conditional name="annotationSource"> + <param name="annotationSourceSelection" type="select" label="Select the type of your annotation"> + <option value="personal_gtf" selected="true">Personal annotation file (GTF format)</option> + <option value="index">Stranded and Unstranded Indexes previously generated by ALFA (Index format)</option> + <option value="built_in_index">Built-in indexes among a list of referenced genome (Index format)</option> + </param> + <when value="personal_gtf"> + <param name="annotationFile" type="data" format="Gff, Gtf" label="Select your personal annotation file (GTF format)"> + </param> + </when> + <when value="index"> + <param name="strandedIndex" type="data" label="Select your ALFA Stranded index file (index format)"/> + <param name="unstrandedIndex" type="data" label="Select your ALFA Unstranded index file (index format)"/> + </when> + <when value="built_in_index"> + <param name="built_in_index_prefix" type="select" label="Select Genome"> + <options from_data_table="alfa_indexes"> + <validator type="no_options" message="No indexes are available for the selected input dataset. Ask your Galaxy Admin for to use ALFA_data_manager tool to build such indexes!" /> + </options> + </param> + </when> + </conditional> + </section> + + <section name="reads" title="INPUT 2: Mapped reads" expanded="True"> + <conditional name="readsType"> + <param name="readsTypeSelection" type="select" label="Select the format of your mapped reads"> + <option value="bam" selected="true">BAM</option> + <option value="bedgraph">BEDGRAPH</option> + </param> + <when value="bam"> + <repeat name="readsList" title="Mapped Reads" min="1" > + <param name="readsFile" type="data" format="Bam" label="Select the file (BAM format)"/> + <param name="readsLabel" type="text" size="20" value="" label="Label of the reads" optional="True"/> + </repeat> + </when> + <when value="bedgraph"> + <repeat name="readsList" title="Mapped Reads" min="1"> + <param name="readsFile" type="data" format="Bed" label="Select the file (BEDGRAPH format)"/> + <param name="readsLabel" type="text" size="20" value="" label="Label of the reads" optional="True"/> + </repeat> + </when> + </conditional> + <param name="strandness" type="select" label="Select the strandness of your library of reads"> + <option value="unstranded" selected="true">Unstranded (reads will be intersected with both forward and reverse strands of the annotated sequence)</option> + <option value="forward">Forward (reads will be intersected with only the the forward strand of the annotated sequence)</option> + <option value="reverse">Reverse (reads will will be intersected only with the reverse strand of the annotated sequence)</option> + </param> + </section> + + <section name="outputFiles" title="OUTPUT FILES: Choose the output files" expanded="False"> + <param name="plot" type="boolean" truevalue="True" falsevalue="False" checked="True" label="Categories and Biotypes Histograms" help="Plot the distribution of genomic categories and biotypes captured by your reads"/> + <param name="countFile" type="boolean" truevalue="True" falsevalue="False" checked="True" label="Categories Count File" help="Return the exact count of nucleotides per genomic categories and biotypes"/> + <param name="index" type="boolean" truevalue="True" falsevalue="False" checked="False" label ="Indexes" help="Return the stranded and unstranded ALFA indexes generated from the GTF input file (useful if you plan to run ALFA again with the same annotated sequence)"/> + </section> + + <section name="outputOptions" title="ADVANCED SETTINGS" expanded="False"> + <param name="categoriesDepth" type="select" label="Categories to Display"> + <option value="1">gene | intergenic | antisense</option> + <option value="2">exon | intron | undescribed genes | intergenic | antisense</option> + <option value="3" selected="true">5’-UTR | CDS | 3’-UTR | underscribes exons | intron | undescribed genes | intergenic | antisense</option> + <option value="4">5’-UTR | start_codon | CDS | undescribed CDS | stop_codon | 3’-UTR | undescribed exons | intron | undescribed genes | intergenic | antisense</option> + </param> + <param name="plotFormat" type="select" label="Plot Options: Select graph format" help="Ignore if you did not choose the histograms output file"> + <option value="png" selected="true">png</option> + <!--<option value="pdf" selected="true">pdf</option>--> + <option value="svg">svg</option> + </param> + <conditional name="plotThreshold"> + <param name="plotThresholdChoice" type="boolean" truevalue="True" falsevalue="False" checked="false" label="Plot Options: Modify y axis range of the normalized counts of bio-features" help="Ignore if you did not choose the histograms output file"/> + <when value="True"> + <param name="yMin" type="float" value="-2.0" label="y min"/> + <param name="yMax" type="float" value="2.0" label="y max"/> + </when> + <when value="False"></when> + </conditional> + </section> + </inputs> + + <outputs> + <data name="logReport" format="txt" label="${projectName}-Log Report"/> + <!-- + <data name="outputPdf" format="pdf" label="${projectName}-BioFeatures Distribution"> + <filter>outputFiles['plot'] is True and outputOptions['plotFormat'] == 'pdf'</filter> + </data> + --> + <data name="outputCategoriesPng" format="png" label="${projectName}-Categories Distribution"> + <filter>outputFiles['plot'] is True and outputOptions['plotFormat'] == 'png'</filter> + </data> + <data name="outputBiotypesPng" format="png" label="${projectName}-Biotypes Distribution"> + <filter>outputFiles['plot'] is True and outputOptions['plotFormat'] == 'png'</filter> + </data> + <data name="outputCategoriesSvg" format="svg" label="${projectName}-Categories Distribution"> + <filter>outputFiles['plot'] is True and outputOptions['plotFormat'] == 'svg'</filter> + </data> + <data name="outputBiotypesSvg" format="svg" label="${projectName}-Biotypes Distribution"> + <filter>outputFiles['plot'] is True and outputOptions['plotFormat'] == 'svg'</filter> + </data> + <data name="outputCountFile" format="txt" label="${projectName}-Categories Count"> + <filter>outputFiles['countFile'] is True</filter> + </data> + <data name="outputStrandedIndex" format="txt" label="${projectName}-Stranded Index"> + <filter>outputFiles['index'] is True</filter> + </data> + <data name="outputUnstrandedIndex" format="txt" label="${projectName}-Unstranded Index"> + <filter>outputFiles['index'] is True</filter> + </data> + </outputs> + + <tests> + <test> + <param name="alfa_toy" /> + <section name="annotation"> + <conditional name="annotationSource"> + <param name="annotationSourceSelection" value="personal_gtf" /> + <param name="annotationFile" value="alfa_toy.gtf" ftype="gtf" /> + </conditional> + </section> + <section name="reads"> + <conditional name="readsType"> + <param name="readsTypeSelection" value="bam" /> + <repeat name="readsList"> + <param name="readsFile" value="alfa_toy.bam" ftype="bam" /> + <param name="readsLabel" value="alfa_toy" /> + </repeat> + <param name="strandness" value="unstranded" /> + </conditional> + </section> + <section name="outputFiles"> + <param name="plot" value="True" /> + <param name="countFile" value="True" /> + <param name="index" value="True" /> + </section> + <section name="outputOptions"> + <param name="categoriesDepth" value="3" /> + <param name="plotFormat" value="pdf" /> + <conditional name="plotThreshold"> + <param name="plotThresholdChoice" value="False" /> + </conditional> + </section> + <output name="outputPdf" file="alfa_toy-Biofeatures Distribution.pdf" ftype="pdf" /> + <output name="outputCountFile" file="alfa_toy.categories_count" ftype="txt" /> + <output name="outputStrandedIndex" file="alfa_toy.stranded.index" ftype="txt" /> + <output name="outputUnstrandedIndex" file="alfa_toy.unstranded.index" ftype="txt" /> + <assert_stdout> + <has_text text="### End of the program" /> + </assert_stdout> + </test> + </tests> + + <help> +<![CDATA[ +**What it does** + + + | ALFA provides a global overview of features distribution composing New Generation Sequencing dataset(s). + | + | Given a set of aligned reads (BAM files) and an annotation file (GTF format), the tool produces plots of the raw and normalized distributions of those reads among genomic categories (stop codon, 5'-UTR, CDS, intergenic, etc.) and biotypes (protein coding genes, miRNA, tRNA, etc.). Whatever the sequencing technique, whatever the organism. + +---- + +**ALFA acronym** + +- Annotation Landscape For Aligned reads + +---- + +**Official documentation of the tool** + + +- https://github.com/biocompibens/ALFA + +---- + +**Detailed example** + +- https://github.com/biocompibens/ALFA#detailed-example + +---- + +**Nota Bene** + +* **Input 1: Annotation File** + + + | ALFA requires as first input an annotation file (sequence, genome...) in gtf format in order to generate alfa indexes needed in a second round of the program. + | Indexes are files which list all the coordinates of the categories (stop codon, 5'-UTR, CDS, intergenic...) and biotypes (protein coding genes, miRNA, tRNA, ...) encountered in the annotated sequence. + | + + .. class:: warningmark + + | Gtf File must be sorted. + | + + .. class:: infomark + + | Generation of indexes from an annotation file might be time consuming (i.e ~10min for the human genome). Thus, ALFA allows the user to submit directly indexes generated in previous runs as inputs for a new run. + | + + .. class:: infomark + + | ALFA also enables the use of built-in indexes to save even more computational time. In order to generate easily these built-in indexes, install the data manager tool `ALFA_data_manager`_ available on the toolshed. + + .. _data_manager_build_alfa_indexes: https://toolshed.g2.bx.psu.edu/view/charles-bernard/data_manager_build_alfa_indexes + +* **Input 2: Reads** + + | ALFA requires as second input a single or a set of mapped reads file(s) in either bam or bedgraph format. The coordinates of the mapped reads will be intersected with the according categories and biotypes mentioned in the indexes. + | The strandness option determines which strand of the annotated sequence will be taken into account during this intersection. + | + + .. class:: warningmark + + | Bam or Bedgraph file(s) must be sorted. + | + + .. class:: warningmark + + | Chromosome names in reads and in annotation file (gtf or indexes) must be the same for the intersection to occur + | + +* **Output files** + + | The result of the intersection is a count file displaying the count of nucleotides in the reads for each genomic categories and biotypes. From this count file, plots of the raw and normalized distributions of the reads among these categories are generated. + | In the output files section, the user can choose what kind of files he/she desires as ALFA output. Categories Count File and Plots are proposed by default. + | + + .. class:: infomark + + | The user can also select the 'indexes' option as output. This option is interesting if you plan to run ALFA again with the same submitted annotation file. *See Nota Bene/Input 1: Annotation File for more information.* + | + + - `How the plots look like`_ + + .. _How the plots look like: https://github.com/biocompibens/ALFA#plots + + | + + - `How they are generated`_ + + .. _How they are generated: https://github.com/biocompibens/ALFA#detailed-example + +---- + +**ALFA Developpers** + + | Benoît Noël and Mathieu Bahin: *compbio team, Institut de Biologie de l'Ecole Normale Supérieure de Paris* + +]]> + </help> + + <citations> + <citation type="bibtex">@MISC{ + author="Benoît Noël and Mathieu Bahin" + title="ALFA: Annotation Landscape For Aligned reads" + crossref="https://github.com/biocompibens/ALFA" + institution="Institut de Biologie de l'Ecole Normale Supérieure de Paris" + } + </citation> + </citations> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/ALFA_wrapper.py Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,193 @@ +#!/usr/bin/python + +import argparse +import logging +import os +import re +import shutil +import subprocess +import sys +import tempfile + +def exit_and_explain(msg): + logging.critical(msg) + sys.exit(msg) + +def cleanup_before_exit(tmp_dir): + if tmp_dir and os.path.exists(tmp_dir): + shutil.rmtree(tmp_dir) + +def get_arg(): + parser = argparse.ArgumentParser() + parser.add_argument('--project_name', dest='project_name', action='store', nargs=1, metavar='project_name', type=str) + #Input 1: Annotation File + parser.add_argument('--index', dest='indexes', action='store', nargs=2, metavar=('stranded_index_filename', 'unstranded_index_filename'), type=str) + parser.add_argument('--bi_index', dest='bi_indexes', action='store', nargs=1, metavar='built_in_indexes_dir_path', type=str ) + parser.add_argument('--annotation', dest='annotation_file', action='store', nargs=1, metavar='annotation_gtf_file', type=str ) + #Input 2: Mapped Reads + parser.add_argument('--reads_format', dest='reads_format', action='store', nargs=1, choices=['bam', 'bedgraph'], metavar='reads_format', type=str) + parser.add_argument('--reads', dest='reads', action='store', nargs='+', metavar=('bam_file1 label1',""), type=str) + parser.add_argument('--strandness', dest='strandness', action='store', nargs=1, default=['unstranded'], choices=['unstranded', 'forward', 'reverse'], metavar='strandness', type=str) + #Output files + parser.add_argument('--output_pdf', dest='output_pdf', action='store', nargs=1, metavar='output_pdf_filename', type=str) + parser.add_argument('--output_svg', dest='output_svg', action='store', nargs=2, metavar=('categories_svg_filename', 'biotypes_svg_filename'), type=str) + parser.add_argument('--output_png', dest='output_png', action='store', nargs=2, metavar=('categories_png_filename', 'biotypes_png_filename'), type=str) + parser.add_argument('--output_count', dest='output_count', action='store', nargs=1, metavar='output_count_filename', type=str) + parser.add_argument('--output_index', dest='output_indexes', action='store', nargs=2, metavar=('output_stranded_index_filename', 'output_unstranded_index_filename'), type=str) + #Output Options + parser.add_argument('--categories_depth', dest='categories_depth', action='store', nargs=1, default=[3], choices=range(1,5), metavar='categories_depth', type=int) + parser.add_argument('--plot_format', dest='plot_format', action='store', nargs=1, choices=['pdf', 'png', 'svg'], metavar='plot_format', type=str) + parser.add_argument('--threshold', dest='threshold', action='store', nargs=2, metavar=('yMin', 'yMax'), type=float) + #Internal variables + parser.add_argument('--log_report', dest='log_report', action='store', nargs=1, metavar='log_filename', type=str) + parser.add_argument('--tool_dir', dest='GALAXY_TOOL_DIR', action='store', nargs=1, metavar='galaxy_tool_dir_path', type=str) + args = parser.parse_args() + return args + +def symlink_user_indexes(stranded_index, unstranded_index, tmp_dir): + index='index' + os.symlink(stranded_index, os.path.join(tmp_dir, index + '.stranded.index')) + os.symlink(unstranded_index, os.path.join(tmp_dir, index + '.unstranded.index')) + return index + +def get_input2_args(reads_list, format, tmp_dir): + n = len(reads_list) + if n%2 != 0: + exit_and_explain('Problem with pairing reads filename and reads label') + if format == 'bam': + input2_args = '--bam' + elif format == 'bedgraph': + input2_args = '--bedgraph' + k = 0 + reads_filenames = [''] * (n/2) + reads_labels = [''] * (n/2) + for i in range(0, n, 2): + curr_filename = reads_list[i].split('__fname__')[1] + # Alfa checks extension so the filename must end either by .bedgraph or by .bam + # We then create a symlink from file.dat to tmp_dir/annotation_n.<format> to avoid the error message + reads_filenames[k] = os.path.join(tmp_dir, 'annotation_' + str(k) + '.' + format) + os.symlink(curr_filename, reads_filenames[k]) + cur_label = reads_list[i+1].split('__label__')[1] + reads_labels[k] = re.sub(r' ', '_', cur_label) + if not reads_labels[k]: + reads_labels[k] = 'sample_%s' % str(k) + input2_args='%s "%s" "%s"' % (input2_args, reads_filenames[k], reads_labels[k]) + k += 1 + return input2_args, reads_filenames, reads_labels + +def redirect_errors(alfa_out, alfa_err): + # When the option --n is enabled, alfa prints '### End of the program' in stderr even if the process worked- + # The following lines to avoid the tool from crashing in this case + if alfa_err and not re.search('### End of program', alfa_err): + # When alfa prints '### End of program' in stdout, all the messages in stderr are considered + # as warnings and not as errors. + if re.search('### End of program', alfa_out): + logging.warning("The script ALFA.py encountered the following warning:\n\n%s" % alfa_err) + logging.info("\n******************************************************************\n") + # True errors make the script exits + else: + exit_and_explain("The script ALFA.py encountered the following error:\n\n%s" % alfa_err) + +def merge_count_files(reads_labels): + merged_count_file = open('count_file.txt', 'wb') + for i in range(0, len(reads_labels)): + current_count_file = open('%s.feature_counts.tsv' % reads_labels[i], 'r') + merged_count_file.write('##LABEL: %s\n\n' % reads_labels[i]) + merged_count_file.write(current_count_file.read()) + merged_count_file.write('__________________________________________________________________\n') + current_count_file.close() + merged_count_file.close() + return 'count_file.txt' + +def main(): + args = get_arg() + + if not (args.output_pdf or args.output_png or args.output_svg or args.output_indexes or args.output_count): + exit_and_explain('Error: no output to return\nProcess Aborted\n') + tmp_dir = tempfile.mkdtemp(prefix='tmp', suffix='') + logging.basicConfig(level=logging.INFO, filename=args.log_report[0], filemode="a+", format='%(message)s') + alfa_path = os.path.join(args.GALAXY_TOOL_DIR[0], 'ALFA.py') + + #INPUT1: Annotation File + if args.indexes: + # The indexes submitted by the user must exhibit the suffix '.(un)stranded.index' and will be called by alfa by their prefix + index = symlink_user_indexes(args.indexes[0], args.indexes[1], tmp_dir) + input1_args = '-g "%s"' % index + elif args.bi_indexes: + input1_args = '-g "%s"' % args.bi_indexes[0] + elif args.annotation_file: + input1_args = '-a "%s"' % args.annotation_file[0] + else: + exit_and_explain('No annotation file submitted !') + + #INPUT 2: Mapped Reads + if args.reads: + input2_args, reads_filenames, reads_labels = get_input2_args(args.reads, args.reads_format[0], tmp_dir) + strandness = '-s %s' % args.strandness[0] + else: + exit_and_explain('No reads submitted !') + + ##Output options + categories_depth = '-d %s' % args.categories_depth[0] + if not (args.output_pdf or args.output_png or args.output_svg): + output_args = '--n' + else: + plot_suffix = os.path.join(tmp_dir, "ALFA_plot"); + if args.output_pdf: + output_args = '--pdf ' + plot_suffix + '.pdf' + if args.output_png: + output_args = '--png ' + plot_suffix + if args.output_svg: + output_args = '--svg ' + plot_suffix + if args.threshold: + output_args = '%s -t %.3f %.3f' % (output_args, args.threshold[0], args.threshold[1]) + + ##Run alfa + cmd = 'python %s %s %s %s %s %s' % (alfa_path, input1_args, input2_args, strandness, categories_depth, output_args) + # Change into the tmp dir because ALFA produces files in the current dir + curr_dir = os.getcwd() + os.chdir(tmp_dir) + print(cmd) + logging.info("__________________________________________________________________\n") + logging.info("Alfa execution") + logging.info("__________________________________________________________________\n") + logging.info("Command Line:\n%s\n" % cmd) + logging.info("------------------------------------------------------------------\n") + alfa_result = subprocess.Popen(args=cmd, shell=True, stdout=subprocess.PIPE, stderr=subprocess.PIPE) + alfa_out, alfa_err = alfa_result.communicate() + + ##Handle stdout, warning, errors... + redirect_errors(alfa_out, alfa_err) + + logging.info("Alfa prompt:\n%s" % alfa_out) + + ##Redirect outputs + if args.output_pdf: + shutil.move(plot_suffix + '.pdf', args.output_pdf[0]) + if args.output_png: + shutil.move(plot_suffix + '.Categories.png', args.output_png[0]) + shutil.move(plot_suffix + '.Biotypes.png', args.output_png[1]) + if args.output_svg: + shutil.move(plot_suffix + '.Categories.svg', args.output_svg[0]) + shutil.move(plot_suffix + '.Biotypes.svg', args.output_svg[1]) + if args.output_count: + count_filename = merge_count_files(reads_labels) + shutil.move(count_filename, args.output_count[0]) + if args.output_indexes: + if args.annotation_file: + indexes_regex = re.compile('.*\.index') + indexes = filter(indexes_regex.search, os.listdir('.')) + indexes.sort() + shutil.move(indexes[0], args.output_indexes[0]) + shutil.move(indexes[1], args.output_indexes[1]) + if args.indexes: + shutil.move(index + '.stranded.index', args.output_indexes[0]) + shutil.move(index + '.unstranded.index', args.output_indexes[1]) + if args.bi_indexes: + shutil.move(args.bi_indexes[0] + '.stranded.index', args.output_index[0]) + shutil.move(args.bi_indexes[1] + '.unstranded.index', args.output_index[1]) + + # Get back to the original dir and cleanup the tmp dir + os.chdir(curr_dir) + cleanup_before_exit(tmp_dir) +main()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/test-data/alfa_toy.bedgraph Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,4 @@ +Chr1 149 199 2 +Chr1 299 349 1 +Chr1 499 549 6 +Chr1 1099 1149 1
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/test-data/alfa_toy.categories_counts Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,5 @@ +#Category,biotype Counts_in_bam Size_in_genome +CDS,protein_coding 300.0 624.0 +five_prime_utr,protein_coding 75.0 250.5 +three_prime_utr,protein_coding 25.0 126.5 +intergenic,intergenic 100.0 249.0
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/test-data/alfa_toy.gtf Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,6 @@ +Chr1 ensembl_havana gene 250 1250 . + . gene_id "ENSMUSG00000051951"; gene_biotype "protein_coding"; +Chr1 ensembl_havana transcript 250 1250 . + . gene_id "ENSMUSG00000051951"; gene_biotype "protein_coding"; +Chr1 ensembl_havana exon 375 1000 . + . gene_id "ENSMUSG00000051951"; gene_biotype "protein_coding"; +Chr1 ensembl_havana CDS 375 1000 . + 0 gene_id "ENSMUSG00000051951"; gene_biotype "protein_coding"; +Chr1 ensembl_havana five_prime_utr 250 375 . - . gene_id "ENSMUSG00000051951"; gene_biotype "protein_coding"; +Chr1 ensembl_havana three_prime_utr 1000 1250 . - . gene_id "ENSMUSG00000051951"; gene_biotype "protein_coding";
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/test-data/alfa_toy.stranded.index Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,11 @@ +#Chr1 1250 +Chr1 249 374 + protein_coding:gene,transcript +Chr1 249 374 - protein_coding:five_prime_utr +Chr1 374 375 + protein_coding:exon,CDS +Chr1 374 375 - protein_coding:five_prime_utr,three_prime_utr +Chr1 375 999 + protein_coding:exon,CDS +Chr1 375 999 - antisense +Chr1 999 1000 + protein_coding:exon,CDS +Chr1 999 1000 - protein_coding:three_prime_utr +Chr1 1000 1250 + protein_coding:gene,transcript +Chr1 1000 1250 - protein_coding:five_prime_utr,three_prime_utr,exon,CDS
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/test-data/alfa_toy.unstranded.index Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,6 @@ +#Chr1 1250 +Chr1 249 374 . protein_coding:five_prime_utr,gene,transcript +Chr1 374 375 . protein_coding:five_prime_utr,three_prime_utr,exon,CDS +Chr1 375 999 . protein_coding:exon,CDS +Chr1 999 1000 . protein_coding:three_prime_utr,exon,CDS +Chr1 1000 1250 . protein_coding:five_prime_utr,exon,CDS,three_prime_utr,gene,transcript
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/tool-data/alfa_indexes.loc.sample Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,2 @@ +#<species> <version> <release> <value> <dbkey> <name> <prefix> +#Dictyostelium_discoideum dicty_2 7 Dictyostelium_discoideum_dicty_2_7 Dictyostelium_discoideum_dicty_2_7 Dictyostelium_discoideum: dicty_2 (release 7) <path_to_dicty_indexes_dir>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/tool_data_table_conf.xml.sample Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,7 @@ +<tables> + <!-- Locations of all alfa indexes --> + <table name="alfa_indexes" comment_char="#" allow_duplicate_entries="False"> + <columns>species, version, release, value, dbkey, name, prefix</columns> + <file path="tool-data/alfa_indexes.loc" /> + </table> +</tables>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/alfa/tool_dependencies.xml Wed May 16 09:49:18 2018 -0400 @@ -0,0 +1,12 @@ +<?xml version="1.0"?> +<tool_dependency> + <package name="bedtools" version="2.24"> + <repository changeset_revision="3416a1d4a582" name="package_bedtools_2_24" owner="iuc" toolshed="https://toolshed.g2.bx.psu.edu" /> + </package> + <package name="samtools" version="1.2"> + <repository changeset_revision="f6ae3ba3f3c1" name="package_samtools_1_2" owner="iuc" toolshed="https://toolshed.g2.bx.psu.edu" /> + </package> + <package name="matplotlib" version="1.4"> + <repository changeset_revision="f7424e1cf115" name="package_python_2_7_matplotlib_1_4" owner="iuc" toolshed="https://toolshed.g2.bx.psu.edu" /> + </package> +</tool_dependency>