File: plotCoverage.py

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#!/usr/bin/python3
# -*- coding: utf-8 -*-

import os
import sys
import argparse
import numpy as np

import matplotlib
matplotlib.use('Agg')
matplotlib.rcParams['pdf.fonttype'] = 42
matplotlib.rcParams['svg.fonttype'] = 'none'
from deeptools import cm  # noqa: F401
import matplotlib.pyplot as plt
import plotly.offline as py
import plotly.graph_objs as go
from importlib.metadata import version
import deeptools.countReadsPerBin as countR
from deeptools import parserCommon
from deeptools.utilities import smartLabels

old_settings = np.seterr(all='ignore')


def parse_arguments(args=None):
    parent_parser = parserCommon.getParentArgParse(binSize=False)
    read_options_parser = parserCommon.read_options()

    parser = \
        argparse.ArgumentParser(
            parents=[required_args(), parent_parser, read_options_parser],
            formatter_class=argparse.RawDescriptionHelpFormatter,
            add_help=False,
            description="""

This tool is useful to assess the sequencing depth of a given sample.
It samples 1 million bp, counts the number of overlapping reads and can report
a histogram that tells you how many bases are covered how many times.
Multiple BAM files are accepted, but they all should correspond to the same genome assembly.

detailed usage help:
 $ plotCoverage  -h

""",
            epilog='example usages:\nplotCoverage '
                   '--bamfiles file1.bam file2.bam -o results.png\n\n'
                   ' \n\n',
            conflict_handler='resolve',
            usage='plotCoverage -b sample1.bam sample2.bam -o coverage.png \n'
                  'help: plotCoverage -h / plotCoverage --help\n')

    parser.add_argument('--version', action='version',
                        version='%(prog)s {}'.format(version('deeptools')))

    return parser


def process_args(args=None):
    args = parse_arguments().parse_args(args)

    if not args.labels:
        if args.smartLabels:
            args.labels = smartLabels(args.bamfiles)
        else:
            args.labels = [os.path.basename(x) for x in args.bamfiles]
    if args.labels and len(args.bamfiles) != len(args.labels):
        sys.exit("The number of labels does not match the number of BAM files.")

    return args


def required_args():
    parser = argparse.ArgumentParser(add_help=False)
    required = parser.add_argument_group('Required arguments')

    required.add_argument('--bamfiles', '-b',
                          metavar='FILE1 FILE2',
                          help='List of indexed BAM files separated by spaces.',
                          nargs='+',
                          required=True)

    optional = parser.add_argument_group('Optional arguments')

    optional.add_argument("--help", "-h", action="help",
                          help="show this help message and exit")

    optional.add_argument('--plotFile', '-o',
                          type=parserCommon.writableFile,
                          help='File name to save the plot to.')

    optional.add_argument('--labels', '-l',
                          metavar='sample1 sample2',
                          help='User defined labels instead of default labels from '
                               'file names. '
                               'Multiple labels have to be separated by spaces, e.g. '
                               '--labels sample1 sample2 sample3',
                          nargs='+')

    optional.add_argument('--smartLabels',
                          action='store_true',
                          help='Instead of manually specifying labels for the input '
                          'BAM files, this causes deepTools to use the file name '
                          'after removing the path and extension.')

    optional.add_argument('--plotTitle', '-T',
                          help='Title of the plot, to be printed on top of '
                          'the generated image. Leave blank for no title. (Default: %(default)s)',
                          default='')

    optional.add_argument('--skipZeros',
                          help='By setting this option, genomic regions '
                          'that have zero or nan values in _all_ samples '
                          'are excluded.',
                          action='store_true',
                          required=False)

    optional.add_argument('--numberOfSamples', '-n',
                          help='Number of 1 bp regions to sample. (Default: %(default)s)',
                          required=False,
                          type=int,
                          default=1000000)

    optional.add_argument('--BED',
                          help='Limits the coverage analysis to '
                          'the regions specified in these files. This overrides --numberOfSamples. '
                          'Due to memory requirements, it is inadvised to combine this with '
                          '--outRawCounts or many tens of thousands of regions, as per-base '
                          'coverage is used!',
                          metavar='FILE1.bed FILE2.bed',
                          nargs='+')

    optional.add_argument('--outRawCounts',
                          help='Save raw counts (coverages) to file.',
                          type=parserCommon.writableFile,
                          metavar='FILE')

    optional.add_argument('--outCoverageMetrics',
                          help='Save percentage of bins/regions above the specified thresholds to '
                          'the specified file. The coverage thresholds are specified by '
                          '--coverageThresholds. If no coverage thresholds are specified, the file '
                          'will be empty.',
                          type=parserCommon.writableFile,
                          metavar='FILE')

    optional.add_argument('--coverageThresholds', '-ct',
                          type=int,
                          action="append",
                          help='The percentage of reported bins/regions with signal at least as '
                          'high as the given threshold. This can be specified multiple times.')

    optional.add_argument('--plotHeight',
                          help='Plot height in cm. (Default: %(default)s)',
                          type=float,
                          default=5.0)

    optional.add_argument('--plotWidth',
                          help='Plot width in cm. The minimum value is 1 cm. (Default: %(default)s)',
                          type=float,
                          default=15.0)

    optional.add_argument('--plotFileFormat',
                          metavar='FILETYPE',
                          help='Image format type. If given, this option '
                          'overrides the image format based on the plotFile '
                          'ending. The available options are: png, '
                          'eps, pdf, svg and plotly.',
                          default=None,
                          choices=['png', 'pdf', 'svg', 'eps', 'plotly'])

    return parser


def main(args=None):
    args = process_args(args)

    if not args.outRawCounts and not args.plotFile and not args.outCoverageMetrics:
        sys.exit("At least one of --plotFile, --outRawCounts and --outCoverageMetrics are required.\n")

    if 'BED' in args:
        bed_regions = args.BED
    else:
        bed_regions = None

    cr = countR.CountReadsPerBin(args.bamfiles,
                                 binLength=1,
                                 bedFile=bed_regions,
                                 numberOfSamples=args.numberOfSamples,
                                 numberOfProcessors=args.numberOfProcessors,
                                 verbose=args.verbose,
                                 region=args.region,
                                 blackListFileName=args.blackListFileName,
                                 extendReads=args.extendReads,
                                 minMappingQuality=args.minMappingQuality,
                                 ignoreDuplicates=args.ignoreDuplicates,
                                 center_read=args.centerReads,
                                 samFlag_include=args.samFlagInclude,
                                 samFlag_exclude=args.samFlagExclude,
                                 minFragmentLength=args.minFragmentLength,
                                 maxFragmentLength=args.maxFragmentLength,
                                 bed_and_bin=True,
                                 out_file_for_raw_data=args.outRawCounts)

    num_reads_per_bin = cr.run()

    if args.outCoverageMetrics and args.coverageThresholds:
        args.coverageThresholds.sort()  # Galaxy in particular tends to give things in a weird order
        of = open(args.outCoverageMetrics, "w")
        of.write("Sample\tThreshold\tPercent\n")
        nbins = float(num_reads_per_bin.shape[0])
        for thresh in args.coverageThresholds:
            vals = np.sum(num_reads_per_bin >= thresh, axis=0)
            for lab, val in zip(args.labels, vals):
                of.write("{}\t{}\t{:6.3f}\n".format(lab, thresh, 100. * val / nbins))
        of.close()

    if args.outRawCounts:
        # append to the generated file the
        # labels
        header = "#plotCoverage --outRawCounts\n#'chr'\t'start'\t'end'\t"
        header += "'" + "'\t'".join(args.labels) + "'\n"
        f = open(args.outRawCounts, 'r+')
        content = f.read()
        f.seek(0, 0)
        f.write(header + content)
        f.close()

    if num_reads_per_bin.shape[0] < 2:
        exit("ERROR: too few non-zero bins found.\n"
             "If using --region please check that this "
             "region is covered by reads.\n")

    if args.skipZeros:
        num_reads_per_bin = countR.remove_row_of_zeros(num_reads_per_bin)

    if args.plotFile:
        if args.plotFileFormat == 'plotly':
            fig = go.Figure()
            fig['layout']['xaxis1'] = {'domain': [0.0, 0.48], 'anchor': 'x1', 'title': 'coverage (#reads per base)'}
            fig['layout']['xaxis2'] = {'domain': [0.52, 1.0], 'anchor': 'x2', 'title': 'coverage (#reads per base)'}
            fig['layout']['yaxis1'] = {'domain': [0.0, 1.0], 'anchor': 'x1', 'title': 'fraction of bases sampled'}
            fig['layout']['yaxis2'] = {'domain': [0.0, 1.0], 'anchor': 'x2', 'title': 'fraction of bases sampled >= coverage'}
            fig['layout'].update(title=args.plotTitle)
        else:
            fig, axs = plt.subplots(1, 2, figsize=(args.plotWidth, args.plotHeight))
            plt.suptitle(args.plotTitle)

    # plot up to two std from mean
    num_reads_per_bin = num_reads_per_bin.astype(int)
    sample_mean = num_reads_per_bin.mean(axis=0)
    sample_std = num_reads_per_bin.std(axis=0)
    sample_max = num_reads_per_bin.max(axis=0)
    sample_min = num_reads_per_bin.min(axis=0)
    sample_25 = np.percentile(num_reads_per_bin, 25, axis=0)
    sample_50 = np.percentile(num_reads_per_bin, 50, axis=0)
    sample_75 = np.percentile(num_reads_per_bin, 75, axis=0)

    # use the largest 99th percentile from all samples to set the x_max value
    x_max = np.max(np.percentile(num_reads_per_bin, 99, axis=0))
    # plot coverage
    # print headers for text output
    print("sample\tmean\tstd\tmin\t25%\t50%\t75%\tmax")
    # the determination of a sensible value for y_max of the first plot (fraction of bases sampled vs.
    # coverage) is important because, depending on the data,
    # it becomes very difficult to see the lines in the plot. For example, if the coverage of a sample
    # is a nice gaussian curve with a large mean of 50. Then a sensible range for the y axis (fraction of
    # reads having coverage=x) is (0, 0.02) which nicely shows the coverage curve. If instead the coverage is
    # very por and centers close to 1 then a good y axis range is (0,1).

    # the current implementation aims to find the y_value for which 50% of the reads >= x (coverage) and
    # sets that as the x_axis range.
    y_max = []
    data = []
    # We need to manually set the line colors so they're shared between the two plots.
    plotly_colors = ["#d73027", "#fc8d59", "#f33090", "#e0f3f8", "#91bfdb", "#4575b4"]
    plotly_styles = sum([6 * ["solid"], 6 * ["dot"], 6 * ["dash"], 6 * ["longdash"], 6 * ["dashdot"], 6 * ["longdashdot"]], [])
    for idx, col in enumerate(num_reads_per_bin.T):
        if args.plotFile:
            frac_reads_per_coverage = np.bincount(col.astype(int)).astype(float) / num_reads_per_bin.shape[0]
            csum = np.bincount(col.astype(int))[::-1].cumsum()
            csum_frac = csum.astype(float)[::-1] / csum.max()
            if args.plotFileFormat == 'plotly':
                color = plotly_colors[idx % len(plotly_colors)]
                dash = plotly_styles[idx % len(plotly_styles)]
                trace = go.Scatter(x=np.arange(0, int(x_max) - 1),
                                   y=frac_reads_per_coverage[:int(x_max)],
                                   mode='lines',
                                   xaxis='x1',
                                   yaxis='y1',
                                   line=dict(color=color, dash=dash),
                                   name="{}, mean={:.1f}".format(args.labels[idx], sample_mean[idx]),
                                   legendgroup="{}".format(idx))
                data.append(trace)
                trace = go.Scatter(x=np.arange(0, int(x_max) - 1),
                                   y=csum_frac[:int(x_max)],
                                   mode='lines',
                                   xaxis='x2',
                                   yaxis='y2',
                                   line=dict(color=color, dash=dash),
                                   name=args.labels[idx],
                                   showlegend=False,
                                   legendgroup="{}".format(idx))
                data.append(trace)
            else:
                axs[0].plot(frac_reads_per_coverage, label="{}, mean={:.1f}".format(args.labels[idx], sample_mean[idx]))
                axs[1].plot(csum_frac, label=args.labels[idx])
            # find the indexes (i.e. the x values) for which the cumulative distribution 'fraction of bases
            # sampled >= coverage' where fraction of bases sampled = 50%: `np.flatnonzero(csum_frac>0.5)`
            # then find the fraction of bases sampled that that have the largest x
            y_max.append(frac_reads_per_coverage[max(np.flatnonzero(csum_frac > 0.5))])
        print("{}\t{:0.2f}\t{:0.2f}\t{}\t{}\t{}\t{}\t{}\t".format(args.labels[idx],
                                                                  sample_mean[idx],
                                                                  sample_std[idx],
                                                                  sample_min[idx],
                                                                  sample_25[idx],
                                                                  sample_50[idx],
                                                                  sample_75[idx],
                                                                  sample_max[idx],
                                                                  ))

    if args.plotFile:
        # Don't clip plots
        y_max = max(y_max)
        if args.plotFileFormat == "plotly":
            fig.add_traces(data)
            fig['layout']['yaxis1'].update(range=[0.0, min(1, y_max + (y_max * 0.10))])
            fig['layout']['yaxis2'].update(range=[0.0, 1.0])
            py.plot(fig, filename=args.plotFile, auto_open=False)
        else:
            axs[0].set_ylim(0, min(1, y_max + (y_max * 0.10)))
            axs[0].set_xlim(0, x_max)
            axs[0].set_xlabel('coverage (#reads per bp)')
            axs[0].legend(fancybox=True, framealpha=0.5)
            axs[0].set_ylabel('fraction of bases sampled')
            # plot cumulative coverage
            axs[1].set_xlim(0, x_max)
            axs[1].set_xlabel('coverage (#reads per bp)')
            axs[1].set_ylabel('fraction of bases sampled >= coverage')
            axs[1].legend(fancybox=True, framealpha=0.5)
            plt.savefig(args.plotFile, format=args.plotFileFormat)
            plt.close()


if __name__ == "__main__":
    main()