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// ==========================================================================
// NGS: Regions of Interest Analysis
// ==========================================================================
// Copyright (c) 2012-2026, Bernd Jagla, Institut Pasteur
// All rights reserved.
//
// Redistribution and use in source and binary forms, with or without
// modification, are permitted provided that the following conditions are met:
//
// * Redistributions of source code must retain the above copyright
// notice, this list of conditions and the following disclaimer.
// * Redistributions in binary form must reproduce the above copyright
// notice, this list of conditions and the following disclaimer in the
// documentation and/or other materials provided with the distribution.
// * Neither the name of Knut Reinert or the FU Berlin nor the names of
// its contributors may be used to endorse or promote products derived
// from this software without specific prior written permission.
//
// THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS"
// AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE
// IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE
// ARE DISCLAIMED. IN NO EVENT SHALL KNUT REINERT OR THE FU BERLIN BE LIABLE
// FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL
// DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR
// SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER
// CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT
// LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY
// OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH
// DAMAGE.
//
// ==========================================================================
// Author: Bernd Jagla <bernd.jagla@pasteur.fr>
// Author: Manuel Holtgrewe <manuel.holtgrewe@fu-berlin.de>
// ==========================================================================
#include <fstream>
#include <iostream>
#include <seqan/arg_parse.h>
#include <seqan/basic.h>
#include <seqan/sequence.h>
#include <seqan/file.h>
#include <seqan/stream.h>
#include <seqan/bam_io.h>
#include "roi_builder.h"
// ==========================================================================
// Classes
// ==========================================================================
// ----------------------------------------------------------------------------
// Class Options
// ----------------------------------------------------------------------------
// Options for the bam2roi Application.
struct Options
{
// Verbosity of output: 0 -- quiet, 1 -- normal, 2 -- verbose, 3 -- very verbose.
int verbosity;
// -----------------------------------------------------------------------
// Input / Output Options
// -----------------------------------------------------------------------
// Paths to input read files.
seqan2::CharString inputFileName;
// Paths to output read files of accepted reads.
seqan2::CharString outputFileName;
// -----------------------------------------------------------------------
// ROI Creation Options
// -----------------------------------------------------------------------
// Whether or not the experiment is strand-specific.
bool strandSpecific;
// Whether or not to use paired information.
bool usePairing;
// Whether or not to link over skipped bases.
bool linkOverSkipped;
Options() : verbosity(0), strandSpecific(false), usePairing(false), linkOverSkipped(false)
{}
};
// ==========================================================================
// Functions
// ==========================================================================
// --------------------------------------------------------------------------
// Function yesNo() [Options]
// --------------------------------------------------------------------------
char const * yesNo(bool b)
{
return b ? "YES" : "NO";
}
// --------------------------------------------------------------------------
// Function print() [Options]
// --------------------------------------------------------------------------
void print(std::ostream & out, Options const & options)
{
out << "__OPTIONS_____________________________________________________________________\n"
<< "\n"
<< "INPUT FILE \t" << options.inputFileName << "\n"
<< "OUTPUT FILE \t" << options.outputFileName << "\n"
<< "\n"
<< "STRAND SPECIFIC \t" << yesNo(options.strandSpecific) << "\n"
<< "USE PAIRED INFO \t" << yesNo(options.usePairing) << "\n"
<< "LINK OVER SKIPPED\t" << yesNo(options.linkOverSkipped) << "\n"
<< "\n";
}
// --------------------------------------------------------------------------
// Function parseCommandLine()
// --------------------------------------------------------------------------
// Parse command line with ArgumentParser class.
seqan2::ArgumentParser::ParseResult
parseCommandLine(Options & options, int argc, char const ** argv)
{
// Setup ArgumentParser.
seqan2::ArgumentParser parser("bam2roi");
setCategory(parser, "NGS ROI Analysis");
// Set short description, version, and date.
setShortDescription(parser, "Create ROI from BAM file.");
setVersion(parser, SEQAN_APP_VERSION " [" SEQAN_REVISION "]");
setDate(parser, SEQAN_DATE);
// Define usage line and long description.
addUsageLine(parser, "\\fB-if\\fP \\fIIN.bam\\fP \\fB-of\\fP \\fIOUT.roi\\fP");
addDescription(parser,
"Calculated consecutive regions of coverage from alignment file \\fIIN.bam\\fP "
"and write regions of interest to file \\fIOUT.roi\\fP. "
"Counting is performed over the entire region (including intron and N-regions) "
"based on the CIGAR string of the alignment record.");
// it is in the scope of this application to make any assumtions over splice junctions
// since there are many boundary issues concerning splice junctions of many reads we
// consider this an alignment artefact. Splice junctions can be analyzed separately by
// using special GFF annotations in conjunction with these roi files.
// -----------------------------------------------------------------------
// General Options
// -----------------------------------------------------------------------
addSection(parser, "General Options");
addOption(parser, seqan2::ArgParseOption("v", "verbose", "Verbose mode."));
addOption(parser, seqan2::ArgParseOption("vv", "very-verbose", "Very verbose mode."));
// -----------------------------------------------------------------------
// Input / Output Options
// -----------------------------------------------------------------------
addSection(parser, "Input / Output Parameters");
addOption(parser, seqan2::ArgParseOption("if", "input-file", "SAM/BAM formatted file. Must be sorted by coordinate.",
seqan2::ArgParseOption::INPUT_FILE));
setValidValues(parser, "input-file", seqan2::BamFileIn::getFileExtensions());
setRequired(parser, "input-file");
addOption(parser, seqan2::ArgParseOption("of", "output-file", "Output file with regions of interest.",
seqan2::ArgParseOption::OUTPUT_FILE));
setValidValues(parser, "output-file", seqan2::RoiFileIn::getFileExtensions());
setRequired(parser, "output-file");
// -----------------------------------------------------------------------
// Optional Parameters
// -----------------------------------------------------------------------
addSection(parser, "ROI Construction Options");
addOption(parser, seqan2::ArgParseOption("ss", "strand-specific",
"Calculate strand-specific ROIs (see section Strand Specificness below."));
addOption(parser, seqan2::ArgParseOption("ip", "ignore-pairing",
"Ignore paired information. Also see Section ROI Creation Details."));
addOption(parser, seqan2::ArgParseOption("ls", "link-over-skipped", "Link over skipped bases in the read alignment."));
// -----------------------------------------------------------------------
// Documentation of ROI Creation Details.
// -----------------------------------------------------------------------
addTextSection(parser, "ROI Creation Details");
addText(parser,
"By default, ROIs are created from single-end data by looking at the alignment only. Matches, mismatches, "
"and deletions from the CIGAR string are counted as consecutive bases with aligned read bases. Skipped bases "
"(CIGAR operation \"N\") are counted as uncovered and do not link two regions.");
addText(parser,
"This behaviour can be changed by specifying \\fB--link-over-skipped\\fP where these skipped bases are not "
"counted as being covered but they link two otherwise not connected ROIs into one with possibly 0 counts in "
"between.");
addText(parser,
"For paired data, we by default link two ROIs that have no interconnecting bases if there is a pair of reads "
"where one mate maps to either ROI. You can switch off this behaviour using \\fP--ignore-pairing\\fP. The "
"forward and reverse strand of the paired read will still be used when using strand specific information.");
// -----------------------------------------------------------------------
// Documentation of ROI file format.
// -----------------------------------------------------------------------
addTextSection(parser, "ROI File Format");
addText(parser,
"The file begins with a sequence of comments, followed by one column header, followed by the records.");
addText(parser, "\\fIComments\\fP start with a single hash (#) and are ignored in analysis.");
addText(parser,
"The \\fIcolumn header\\fP starts with two hashes (##) and is followed by the tab separated column names. "
"The column names must not contain spaces.");
addText(parser,
"The columns \\fI1-7\\fP are fixed. The last column is always \"counts\". The columns are as follows");
addListItem(parser, "1. ref", "The reference name.");
addListItem(parser, "2. begin_pos", "The begin position.");
addListItem(parser, "3. end_pos", "The end position.");
addListItem(parser, "4. region_name", "The name of the region.");
addListItem(parser, "5. length", "The length of the region.");
addListItem(parser, "6. strand", "The strand, one of \"+\" and \"-\". \"+\" in case of being not strand-specific.");
addListItem(parser, "7. max_count", "The largest value of the \\fIcounts\\fP columns.");
addListItem(parser, "additional columns", "Additional annotation data for the ROI.");
addListItem(parser, "N. counts", "Comma-separated list of \\fIlength\\fP unsignd integers.");
// TODO(holtgrew): Write me!
// -----------------------------------------------------------------------
// Parsing and Value Extraction
// -----------------------------------------------------------------------
// Parse command line.
seqan2::ArgumentParser::ParseResult res = seqan2::parse(parser, argc, argv);
// Only extract options if the program will continue after parseCommandLine()
if (res != seqan2::ArgumentParser::PARSE_OK)
return res;
// Extract option values.
options.verbosity = 1;
if (isSet(parser, "verbose"))
options.verbosity = 2;
if (isSet(parser, "very-verbose"))
options.verbosity = 3;
getOptionValue(options.inputFileName, parser, "input-file");
getOptionValue(options.outputFileName, parser, "output-file");
options.strandSpecific = isSet(parser, "strand-specific");
options.usePairing = !isSet(parser, "ignore-pairing");
options.linkOverSkipped = isSet(parser, "link-over-skipped");
return seqan2::ArgumentParser::PARSE_OK;
}
// --------------------------------------------------------------------------
// Function main()
// --------------------------------------------------------------------------
int main(int argc, char const ** argv)
{
// Parse the command line.
Options options;
seqan2::ArgumentParser::ParseResult res = parseCommandLine(options, argc, argv);
// If parsing was not successful then exit with code 1 if there were errors. Otherwise, exit with code 0 (e.g. help
// was printed).
if (res != seqan2::ArgumentParser::PARSE_OK)
return res == seqan2::ArgumentParser::PARSE_ERROR;
// Print program name and options.
if (options.verbosity >= 1)
{
std::cerr << "BAM TO ROI\n"
<< "==========\n"
<< "\n";
print(std::cerr, options);
}
// -----------------------------------------------------------------------
// Open input and output file
// -----------------------------------------------------------------------
if (options.verbosity >= 1)
std::cerr << "__OPENING FILES_______________________________________________________________\n"
<< "\n";
if (options.verbosity >= 1)
std::cerr << "Opening " << options.inputFileName << " ...";
seqan2::BamFileIn bamFileIn;
if (!open(bamFileIn, toCString(options.inputFileName)))
{
std::cerr << "ERROR: Could not open " << options.inputFileName << "\n";
return 1;
}
if (options.verbosity >= 1)
std::cerr << "OK\n";
if (options.verbosity >= 1)
std::cerr << "Opening " << options.outputFileName << " ...";
seqan2::RoiFileOut roiFileOut;
if (!open(roiFileOut, toCString(options.outputFileName)))
{
std::cerr << "ERROR: Could not open " << options.outputFileName << "\n";
return 1;
}
if (options.verbosity >= 1)
std::cerr << "OK\n";
// -----------------------------------------------------------------------
// Build ROIs
// -----------------------------------------------------------------------
if (options.verbosity >= 1)
std::cerr << "__BUILD ROI___________________________________________________________________\n"
<< "\n"
<< "Working ...";
// We have two RoiBuilder objects, one for ROIs on the forward strand and one for ROIs on the reverse strand. In
// the case of not being strand specific, we only use the forward builder.
RoiBuilderOptions roiBuilderOptions(options.verbosity, options.strandSpecific,
options.usePairing, options.linkOverSkipped);
RoiBuilder roiBuilderF(roiFileOut, roiBuilderOptions);
roiBuilderF.writeHeader(); // only once
RoiBuilder roiBuilderR(roiFileOut, roiBuilderOptions);
// Set the reference sequence names.
seqan2::BamHeader header;
readHeader(header, bamFileIn);
for (unsigned i = 0; i < length(contigNames(context(bamFileIn))); ++i)
{
appendValue(roiBuilderF.refNames, contigNames(context(bamFileIn))[i]);
appendValue(roiBuilderR.refNames, contigNames(context(bamFileIn))[i]);
}
// TODO(holtgrew): This is only suited for the Illumina mate pair protocol at the moment (--> <--).
int oldRId = 0;
int oldPos = 0;
seqan2::BamAlignmentRecord record;
while (!atEnd(bamFileIn))
{
readRecord(record, bamFileIn);
// Break if record is unmapped.
if (hasFlagUnmapped(record))
break; // Found first unmapped record!
// Check sorting.
if (record.rID < oldRId || (record.rID == oldRId && record.beginPos < oldPos))
{
std::cerr << "\nERROR: The BAM file is not sorted properly!\n";
return 1;
}
oldRId = record.rID;
oldPos = record.beginPos;
// Process record, different cases, depending on strandedness and pairedness.
if (hasFlagMultiple(record))
{
if (hasFlagFirst(record) && !hasFlagRC(record))
roiBuilderF.pushRecord(record);
else if (hasFlagFirst(record) && hasFlagRC(record))
roiBuilderR.pushRecord(record);
else if (hasFlagLast(record) && !hasFlagRC(record))
roiBuilderF.pushRecord(record);
else // (hasFlagLast(record) && hasFlagRC(record))
roiBuilderR.pushRecord(record);
}
else
{
if (options.strandSpecific)
{
if (hasFlagRC(record))
roiBuilderR.pushRecord(record);
else
roiBuilderF.pushRecord(record);
}
else
{
roiBuilderF.pushRecord(record);
}
}
}
if (options.verbosity >= 1)
std::cerr << " OK\n"
<< "\n"
<< "Done.\n";
return 0;
}
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