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Read.py
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Read.py
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#!/usr/bin/python
"""Read class for metagene_counts.py.
Requires:
python 2 (https://www.python.org/downloads/)
Based on Perl code by Karl F. Erhard, Jr Copyright (c) 2011
Extended and modified to Python by Joy-El R.B. Talbot Copyright (c) 2014
The MIT License (MIT)
Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in
all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN
THE SOFTWARE.
"""
import re
import subprocess
from MetageneError import MetageneError
from metageneMethods import confirm_integer
from metageneMethods import run_pipe
##TODO: add support for different alignment times (eg. bigwig or bigbed?)
class Read(object):
"""Create a read represented by a list of chromosomal positions.
Class Attributes:
has_sam_tag -- file level information about SAM tags
value: dictionary; key = two-letter SAM tag (eg. "NA", "NH")
value = boolean
cigar_codes -- description of CIGAR alignment options
value: dictionary; key = single character CIGAR code
value = (boolean, boolean) for (counting, advancing position)
reference: Descriptions from Sequence Alignment/Map Format Secification,
28 Feb 2014; version 7fd84b0 from https://github.com/samtools/hts-specs
Attributes:
chromosome -- chromosome
value: matches a key in the chromosome_conversion dict
strand -- strand relative to the chromosome
value: '+' | '-' | '.'
position_array -- chromosome positions (1-based) covered by the read
value: array of integers from 5' to 3' relative to the read itself
with gaps in the read represented as jumps in the positions
start of read (5'-most base) = position_array[0]
end of read (3'-most base) = position_array[-1]
abundance -- read count represented by the alignment line
value: non-zero positive integer; 1 or extracted from NA:i:## tag
mappings -- count of potentially alignment positions
value: non-zero positive integer; 1 or extracted from NH:i:## tag
Class Methods:
create_from_sam -- create read object from SAM/BAM line
parse_sam_bitwise_flag -- return countable and reverse_complement booleans
build_positions -- build positions array from CIGAR alignment
set_sam_tag -- add key:value pairs to has_sam_tag class dictionary
set_chromosome_sizes -- create dictionary of chromosome sizes
"""
__slots__ = ['chromosome', 'strand', 'position_array', 'abundance', 'mappings']
chromosome_sizes = {}
# Keeps track of presence/absence of certain SAM file tags
# (defined by get_sam_tag classmethod)
# Key = two-letter code; value = boolean
has_sam_tag = {}
# How to count[0] and advance[1] chromosome position by CIGAR code
cigar_codes = {'M': (True, True), # alignment match (can be either sequence match or mismatch)
'I': (False, False), # insertion to the reference
'D': (False, True), # deletion from the reference
'N': (False, True), # skipped region from the reference
'S': (False, False), # soft clipping (clipped sequences present in SEQ)
'H': (False, False), # hard clipping (clipped sequences NOT present in SEQ)
'P': (False, True), # padding (silent deletion from padded reference)
'=': (True, True), # sequence match
'X': (True, True)} # sequence mismatch
def __init__(self, chromosome, strand, abundance, mappings, positions):
"""Create read object. Invoke with a constructor rather than directly.
Keyword Arguments:
chromosome -- reference sequence name, often a chromosome
strand -- read strand relative to the reference ['+'|'-'|'.']
abundance -- identical reads represented by the same alignment
mappings -- alignment positions for this read
positions -- array of 1-based chromosome positions the read covers """
self.chromosome = chromosome
if strand != "+" and strand != "-":
self.strand = "."
else:
self.strand = strand
if self.strand == "-":
positions.reverse()
self.position_array = positions
if confirm_integer(abundance, "Abundance", minimum=1):
self.abundance = int(abundance)
if mappings == "Unknown":
self.mappings = 1
elif confirm_integer(mappings, "Alignments", minimum=1):
self.mappings = int(mappings)
def __str__(self):
return "Read at {0}:{1}-{2} on {3} strand; counts for {4:2.3f}:\t\t{5}".format(
self.chromosome,
self.position_array[0], # Start 1-based
self.position_array[-1], # End 1-based
self.strand,
float(self.abundance) / self.mappings,
str(self.position_array))
@classmethod
def create_from_sam(cls,
sam_line,
chromosomes_to_process,
count_method,
unique=False,
ignore_strand=False,
count_secondary_alignments=True,
count_failed_quality_control=False,
count_PCR_optical_duplicate=False,
count_supplementary_alignment=True):
"""Create a Read object from a BAM or SAM line.
Keyword Arguments:
sam_line -- raw line from SAM file or 'samtools view BAM_file' output
chromosome_conversion -- dictionary of chromosome names
count_method -- how to count the read ['all'|'start'|'end']
unique -- boolean for mapping; if True mappings = 1 (default False)
count_secondary_alignments -- process secondary alignment reads (default True)
count_failed_quality_control -- process failed quality control reads (default False)
count_PCR_optical_duplicate -- process PCR/optical duplicate reads (default False)
count_supplementary_alignment -- process supplementary alignment reads (default True)
"""
sam_parts = sam_line.split("\t")
if count_method == 'start':
count_only_start = True
count_only_end = False
elif count_method == 'end':
count_only_start = False
count_only_end = True
else:
count_only_start = False
count_only_end = False
# parse bitwise flag
(countable, reverse_complement) = Read.parse_sam_bitwise_flag(int(sam_parts[1]),
count_secondary_alignments,
count_failed_quality_control,
count_PCR_optical_duplicate,
count_supplementary_alignment,
count_only_start,
count_only_end)
if countable and sam_parts[2] in chromosomes_to_process:
# assign chromosome
chromosome = sam_parts[2]
# assign mappings
if unique:
mappings = 1
# try to extract mappings from NH:i:## tag
elif 'NH' in cls.has_sam_tag and cls.has_sam_tag['NH']:
try:
mappings = int(re.search('NH:i:(\d+)', sam_line).group(1))
except AttributeError:
raise MetageneError("Could not determine number of mappings")
else:
mappings = "Unknown"
# assign abundance either from NA:i:## tag or as 1 (default)
if 'NA' in cls.has_sam_tag and cls.has_sam_tag['NA']:
try:
abundance = int(re.search('NA:i:(\d+)', sam_line).group(1))
except AttributeError:
raise MetageneError("Could not extract the abundance tag")
else:
abundance = 1
# assign strand and positions
if ignore_strand:
strand = "."
else:
if reverse_complement: # Crick or Minus strand
strand = "-"
else: # Watson or Plus strand
strand = "+"
# create genomic positions for read (start, cigar_string, sequence)
positions = Read.build_positions(int(sam_parts[3]), sam_parts[5], sam_parts[9])
return (countable, Read(chromosome, strand, abundance, mappings, positions))
else:
return (False, "Non-aligning read")
# end of create_from_sam
@classmethod
def parse_sam_bitwise_flag(cls,
flags,
count_secondary_alignments=True,
count_failed_quality_control=False,
count_PCR_optical_duplicate=False,
count_supplementary_alignments=True,
count_only_start=False,
count_only_end=False):
"""Parse bitwise flag and return (countable, reverse_complemented) booleans.
Keyword Arguments:
flags -- decimal number representing bitwise flag
count_secondary_alignments -- count reads with bitwise flag 0x100 (default True)
count_failed_quality_control -- count reads with bitwise flag 0x200 (default False)
count_PCR_optical_duplicate -- count reads with bitwise flag 0x400 (default False)
count_supplementary_alignment -- count reads with bitwise flag 0x800 (default True)
count_only_start -- count only alignment start reads; bitwise flag 0x40 (default False)
count_only_end -- count only alignment end reads; bitwise flag 0x80 (default False)
Understanding the SAM/BAM bitwise flag:
Based on description from Sequence Alignment/Map Format Secification,
28 Feb 2014; version 7fd84b0 from https://github.com/samtools/hts-specs
Bit(hex) binary_representation
0x1 0000 0000 0001 template in multiple segments; if 0, MUST ignore string[-2,-4,-6,-7,-8]
0x2 0000 0000 0010 each segment is properly aligned (according to aligner)
0x4 0000 0000 0100 unmapped flag read MUST ignore string[-2,-5,-9,-12] and prev_string[-6]
0x8 0000 0000 1000 next segment in template is unmapped MUST ignore string[-6]
0x10 0000 0001 0000 reverse complement of seq (- strand)
0x20 0000 0010 0000 next segment is reverse complemented
0x40 0000 0100 0000 first segment of template
0x80 0000 1000 0000 last segment of template
*** User input can flip these default behaviors... ***
0x100 0001 0000 0000 secondary alignment
0x200 0010 0000 0000 not passing quality controls
0x400 0100 0000 0000 PCR or optical duplicate
0x800 1000 0000 0000 supplementary alignment
"""
# make sure that only one of count_only_start or count_only_end is true
if count_only_start and count_only_end:
raise MetageneError("You can not count only the start and only the end, choose one or neither")
if (flags & 0x10) == 0:
reverse_complement = False
else:
reverse_complement = True
# Is the read countable?
# Does it map?
if (flags & 0x4) == 0x4: # flag is set and read is unmapped
return (False, reverse_complement)
# Is it a secondary alignment and do we care?
elif (flags & 0x100) == 0x100 and not count_secondary_alignments:
return (False, reverse_complement)
# Did it fail the quality control and do we care?
elif (flags & 0x200) == 0x200 and not count_failed_quality_control:
return (False, reverse_complement)
# Is it a PCR or optical duplicate and do we care?
elif (flags & 0x400) == 0x400 and not count_PCR_optical_duplicate:
return (False, reverse_complement)
# Is it a supplementary alignment and do we care?
elif (flags & 0x800) == 0x800 and not count_supplementary_alignments:
return (False, reverse_complement)
# Do we care about counting only the start or end? and does it matter (because part of a multi-segment template)?
elif (count_only_start or count_only_end) and (flags & 0x1) == 0x1:
# Do we care about the start and does this segment contain the start?
if count_only_start and (flags & 0x40) == 0x40:
return (True, reverse_complement)
# Do we care about the end and does this segment contain the end?
elif count_only_end and (flags & 0x80) == 0x80:
return (True, reverse_complement)
else:
return (False, reverse_complement)
else:
# Made it through everything that could negate counting the read, so count it!
return (True, reverse_complement)
@classmethod
def build_positions(cls, start, cigar, seq):
"""Return array of 1-based positions ordered relative to the chromosome.
Keyword Arguments:
start -- start of read relative to the chromosome: 1-based, left-most position
cigar -- string representation of alignment (discussed below)
seq -- sequence of the read
"""
array = []
position = int(start)
# sometime the cigar value is "*", in which case assume a perfect match
if cigar == "*":
if seq != "*":
for i in range(len(seq)):
array.append(position)
position += 1
return array
else:
raise MetageneError("Unable to determine alignment length")
# separate CIGAR string into nucleotide counts and CIGAR codes
nucleotides = re.findall('(\d+)', cigar)
codes = re.split('\d+', cigar)[1:]
# loop through nucleotide values
for i in range(len(nucleotides)):
# iterate nt times adding 1 to start each time
for j in range(int(nucleotides[i])):
try:
if cls.cigar_codes[codes[i]][0]:
array.append(position)
except KeyError:
raise MetageneError("Incorrect CIGAR string")
if cls.cigar_codes[codes[i]][1]:
position += 1
return array
# end of build_positions
@classmethod
def set_sam_tag(cls, count_tag, bamfile_name, tag_regex):
"""Add key:value pair to class variable: has_sam_tag.
Keyword Arguments:
count_tag -- boolean on whether to count with this tag
bamfile_name -- file to query for tag
tag_regex -- regular expression for the tag (eg 'NA:i:(\d+)')
"""
(run_pipe_worked, sam_sample) = run_pipe(['samtools view {}'.format(bamfile_name), 'head -n 10'])
if run_pipe_worked:
return cls.process_set_sam_tag(sam_sample, count_tag, tag_regex)
else:
raise MetageneError("Checking the bam file failed with error: {}".format(sam_sample))
@classmethod
def process_set_sam_tag(cls, sample, count_tag, tag_regex):
"""Process sample from set_sam_tag. (Separate file handling from processing.)"""
tag = tag_regex.split(":")[0]
num_tags = 0
for sam_line in sample:
if re.search(tag_regex, sam_line) is not None:
num_tags += 1
if num_tags == 10:
has_sam_value = True
else:
has_sam_value = False
if count_tag:
raise MetageneError("Your alignment file does not have the required {} tag.".format(tag))
cls.has_sam_tag[tag] = has_sam_value
return True
@classmethod
def set_chromosome_sizes(cls, bamfile):
"""Set chromosome_sizes dictionary with BAM header.
Keyword Arguments:
bamfile -- name of bamfile
"""
(run_pipe_worked, header) = run_pipe(["samtools view -H {}".format(bamfile)])
if not run_pipe_worked:
raise MetageneError("Could not open BAM file {}".format(bamfile))
else:
try:
return cls.extract_chromosome_sizes(header)
except MetageneError as err:
raise MetageneError("Error processing {} header\n{}".format(bamfile, err.message))
@classmethod
def extract_chromosome_sizes(cls, header):
"""Process header from set_chromosome_sizes. (Separate file handling from processing.)"""
for line in header:
if line[0:3] == "@SQ":
# parse out chromosome information from @SQ lines
name = re.findall('SN:(\S+)', line)[0]
size = int(re.findall('LN:(\d+)', line)[0])
cls.chromosome_sizes[name] = size
if len(cls.chromosome_sizes.keys()) == 0:
raise MetageneError("Could not extract any reference sequence (@SQ) lines from header")
return True
# end of Read class