def assemble_amplicons(contigs_fa=None,
ref_fa=None,
ref_gtf=None,
outdir='.',
sample_id='sampleXX',
padding=50,
min_contig_len=200,
keep_tmp=False,
quiet=False,
logfile=None,
debug=False):
""" Pipeline step to assemble contigs using reference and amplicon regions
Args:
contigs_fa (str): Path to fasta file with assembled contigs
ref_fa (str): Path to reference fasta file
ref_gtf (str): Path to reference GTF file with amplicons
outdir (str): Path to output directory
sample_id (str): Name to append to scaffold sequence
padding (int): Bases to include outside reference annotation
min_contig_len (int): Minimum contig length for tiling path
keep_tmp (bool): Do not delete temporary directory
quiet (bool): Do not write output to console
logfile (file): Append console output to this file
debug (bool): Print commands but do not run
Returns:
out_assembly (str): Path to assembled amplicons (FASTA)
out_summary (str): Path to assembly summary
out_padded (str): Path to padded output file
"""
# Check dependencies
sysutils.check_dependency('nucmer')
sysutils.check_dependency('delta-filter')
sysutils.check_dependency('show-tiling')
# Outputs
out_assembly = os.path.join(outdir, 'amplicon_assembly.fna')
out_summary = os.path.join(outdir, 'amplicon_summary.txt')
out_padded = os.path.join(outdir, 'amplicon_padded.out')
if os.path.exists(out_padded): os.unlink(out_padded)
# Temporary directory
tempdir = sysutils.create_tempdir('assemble_amplicons', None, quiet,
logfile)
# Create fasta file with sequence IDs only (remove decription)
tmp_contigs_fa = sequtils.clean_seqnames_file(contigs_fa, tempdir)
# Load reference sequence(s)
refseqs = {s.id: s for s in SeqIO.parse(ref_fa, 'fasta')}
# For each amplicon, extract the sequence from the reference and scaffold using nucmer
amplicon_alignments = []
amps = [
gl for gl in gtfparse.gtf_parser(ref_gtf) if gl.feature == 'amplicon'
]
for gl in amps:
msg = 'Amplicon ref|%s|reg|%s\n' % (gl.chrom, gl.attrs['name'])
sysutils.log_message(msg, quiet, logfile)
# Extract reference amplicon
amp_s = max(0, (gl.start - 1) - padding)
amp_e = min(len(refseqs[gl.chrom]), gl.end + padding)
ampseq = refseqs[gl.chrom].seq[amp_s:amp_e]
amplicon_fa = os.path.join(tempdir, 'subject.fa')
with open(amplicon_fa, 'w') as outh:
print('>ref|%s|reg|%s' % (gl.chrom, gl.attrs['name']), file=outh)
print(sequtils.wrap(str(ampseq)), file=outh)
# Align with nucmer
fil, til = alignutils.align_nucmer(tmp_contigs_fa,
amplicon_fa,
tempdir,
min_contig_len=min_contig_len,
quiet=quiet,
logfile=logfile,
debug=debug)
# Skip everything else if debugging
if debug: continue
# Parse tiling and show alignments
trows = [alignutils.TilingRow(l) for l in open(til, 'rU')]
if not trows:
amplicon_alignments.append((gl.chrom, gl.attrs['name'], None))
else:
# Initialize alignment
amp_seq = SeqIO.read(amplicon_fa, 'fasta')
combined = alignutils.EmptyReferenceAlignment(
str(amp_seq.seq).lower())
for tr in trows:
out = alignutils.show_aligns(tr.ref, tr.qry, fil)
for nucaln in alignutils.parse_show_aligns(out):
combined = combined.merge_alignments(nucaln)
with open(out_padded, 'a') as outh:
print('%s\n%s\n%s' %
(tr, combined.raln(), combined.qaln()),
file=outh)
amplicon_alignments.append((gl.chrom, gl.attrs['name'], combined))
# Cleanup
for f in [fil, til, amplicon_fa]:
if os.path.isfile(f):
os.unlink(f)
# Write to output files
with open(out_assembly, 'w') as outseq, open(out_summary, 'w') as outsum:
for ref_id, reg, combined in amplicon_alignments:
amp_id = sequtils.make_seq_id(sid=sample_id, ref=ref_id, reg=reg)
if combined is None:
msg1 = '%s\tFAIL\t%d' % (amp_id, 0)
msg2 = u'%s\tFAIL\t%d\t%s\n' % (amp_id, 0, u"👎🏼")
if logfile is not None:
print(u'%s\tFAIL\t%d\t%s' % (amp_id, 0, u"👎🏼"),
file=logfile)
else:
scaf, s, e = combined.scaffold2()
msg1 = '%s\tPASS\t%d' % (amp_id, len(scaf))
msg2 = u'%s\tPASS\t%d\t%s\n' % (amp_id, len(scaf), u"👍🏼")
print('>%s' % (amp_id), file=outseq)
print('%s' % sequtils.wrap(scaf), file=outseq)
print(msg1, file=outsum)
sysutils.log_message(msg2, quiet, logfile)
if not keep_tmp:
sysutils.remove_tempdir(tempdir, 'assemble_amplicons', quiet, logfile)
return out_assembly, out_summary, out_padded
def align_reads(
fq1=None,
fq2=None,
fqU=None,
ref_fa=None,
outdir='.',
bt2_preset='sensitive-local',
sample_id='sampleXX',
no_realign=False,
remove_duplicates=False,
encoding=None,
ncpu=1,
xmx=sysutils.get_java_heap_size(),
keep_tmp=False,
quiet=False,
logfile=None,
debug=False,
):
""" Pipeline step to align reads
Args:
fq1 (str): Path to fastq file with read 1
fq2 (str): Path to fastq file with read 2
fqU (str): Path to fastq file with unpaired reads
ref_fa (str): Path to reference fasta file
outdir (str): Path to output directory
bt2_preset (str): Bowtie2 preset to use for alignment
sample_id (str): Read group ID
no_realign (bool): Do not realign indels
remove_duplicates (bool): Remove duplicates from final alignment
encoding (str): Quality score encoding
ncpu (int): Number of CPUs to use
xmx (int): Maximum heap size for JVM in GB
keep_tmp (bool): Do not delete temporary directory
quiet (bool): Do not write output to console
logfile (file): Append console output to this file
debug (bool): Print commands but do not run
Returns:
out_aligned (str): Path to aligned BAM file
out_bt2 (str): Path to bowtie2 report
"""
# Check inputs
if fq1 is not None and fq2 is not None and fqU is None:
input_reads = "paired" # Paired end
elif fq1 is None and fq2 is None and fqU is not None:
input_reads = "single" # Single end
elif fq1 is not None and fq2 is not None and fqU is not None:
input_reads = "both"
else:
msg = "incorrect input reads; requires either "
msg += "(--fq1 AND --fq2) OR (--fqU) OR (--fq1 AND --fq2 AND --fqU)"
raise MissingRequiredArgument(msg)
if encoding is None:
if input_reads == 'single':
encoding = helpers.guess_encoding(fqU)
else:
encoding = helpers.guess_encoding(fq1)
# Check dependencies
sysutils.check_dependency('bowtie2')
sysutils.check_dependency('samtools')
sysutils.check_dependency('picard')
# Identify correct command for GATK
GATK_BIN = sysutils.determine_dependency_path(['gatk', 'gatk3'])
# Set JVM heap argument (for GATK)
JAVA_HEAP = '_JAVA_OPTIONS="-Xmx%dg"' % xmx
# Outputs
out_aligned = os.path.join(outdir, 'aligned.bam')
out_bt2 = os.path.join(outdir, 'aligned.bt2.out')
# Temporary directory
tempdir = sysutils.create_tempdir('align_reads', None, quiet, logfile)
# Copy and index initial reference
curref = os.path.join(tempdir, 'initial.fasta')
cmd1 = ['cp', ref_fa, curref]
cmd2 = ['samtools', 'faidx', curref]
cmd3 = [
'picard', 'CreateSequenceDictionary',
'R=%s' % curref,
'O=%s' % os.path.join(tempdir, 'initial.dict')
]
cmd4 = ['bowtie2-build', curref, os.path.join(tempdir, 'initial')]
sysutils.command_runner([cmd1, cmd2, cmd3, cmd4], 'align_reads:index',
quiet, logfile, debug)
# Align with bowtie2
cmd5 = [
'bowtie2',
'-p',
'%d' % ncpu,
'--phred33' if encoding == "Phred+33" else '--phred64',
'--no-unal',
'--rg-id',
sample_id,
'--rg',
'SM:%s' % sample_id,
'--rg',
'LB:1',
'--rg',
'PU:1',
'--rg',
'PL:illumina',
'--%s' % bt2_preset,
'-x',
'%s' % os.path.join(tempdir, 'initial'),
]
if input_reads in [
'paired',
'both',
]:
cmd5 += [
'-1',
fq1,
'-2',
fq2,
]
elif input_reads in [
'single',
'both',
]:
cmd5 += [
'-U',
fqU,
]
cmd5 += [
'-S',
os.path.join(tempdir, 'aligned.bt2.sam'),
]
cmd5 += [
'2>',
out_bt2,
]
try:
sysutils.command_runner([
cmd5,
], 'align_reads:bowtie2', quiet, logfile, debug)
except PipelineStepError as e:
if os.path.exists(out_bt2):
with open(out_bt2, 'r') as fh:
print('[--- bowtie2 stderr ---]\n%s' % fh.read(),
file=sys.stderr)
raise
cmd6 = [
'samtools',
'view',
'-u',
os.path.join(tempdir, 'aligned.bt2.sam'),
'|',
'samtools',
'sort',
'>',
os.path.join(tempdir, 'sorted.bam'),
]
cmd7 = [
'samtools',
'index',
os.path.join(tempdir, 'sorted.bam'),
]
sysutils.command_runner([
cmd6,
cmd7,
], 'align_reads:samsort', quiet, logfile, debug)
cur_bam = os.path.join(tempdir, 'sorted.bam')
if remove_duplicates:
sysutils.log_message('[--- Removing duplicates ---]', quiet, logfile)
else:
sysutils.log_message('[--- Marking duplicates ---]', quiet, logfile)
# MarkDuplicates
cmd8 = [
'picard',
'MarkDuplicates',
'CREATE_INDEX=true',
'USE_JDK_DEFLATER=true',
'USE_JDK_INFLATER=true',
'M=%s' % os.path.join(tempdir, 'rmdup.metrics.txt'),
'I=%s' % cur_bam,
'O=%s' % os.path.join(tempdir, 'rmdup.bam'),
]
if remove_duplicates:
cmd8 += [
'REMOVE_DUPLICATES=true',
]
sysutils.command_runner([
cmd8,
], 'align_reads:markdups', quiet, logfile, debug)
cur_bam = os.path.join(tempdir, 'rmdup.bam')
if no_realign:
print('[--- Skipping realignment ---]', file=sys.stderr)
else:
# RealignerTargetCreator
cmd9 = [
JAVA_HEAP,
GATK_BIN,
'-T',
'RealignerTargetCreator',
'-I',
cur_bam,
'-R',
curref,
'-o',
os.path.join(tempdir, 'tmp.intervals'),
]
# IndelRealigner
cmd10 = [
JAVA_HEAP, GATK_BIN, '-T', 'IndelRealigner', '--use_jdk_deflater',
'--use_jdk_inflater', '-maxReads', '1000000', '-dt', 'NONE', '-I',
cur_bam, '-R', curref, '-targetIntervals',
os.path.join(tempdir, 'tmp.intervals'), '-o',
os.path.join(tempdir, 'realign.bam')
]
sysutils.command_runner([
cmd9,
cmd10,
], 'align_reads:realign', quiet, logfile, debug)
cur_bam = os.path.join(tempdir, 'realign.bam')
# Check that cur_bam was created
if not os.path.exists(cur_bam):
msg = "BAM does not exist: %s" % cur_bam
raise sysutils.PipelineStepError(msg)
cmd11a = [
'rm',
'-f',
out_aligned,
]
cmd11b = [
'mv',
cur_bam,
out_aligned,
]
cmd11c = [
'samtools',
'index',
out_aligned,
]
sysutils.command_runner([
cmd11a,
cmd11b,
cmd11c,
], 'align_reads:copy', quiet, logfile, debug)
if not keep_tmp:
sysutils.remove_tempdir(tempdir, 'align_reads', quiet, logfile)
return out_aligned, out_bt2
def vcf_to_consensus(
vcf=None,
outdir='.',
sampidx=0,
min_dp=5,
major=0.5,
minor=0.2,
keep_tmp=False,
quiet=False,
logfile=None,
):
""" Pipeline step to create consensus sequence from VCF
Args:
vcf (str): Path to variant calls (VCF)
outdir (str): Path to output directory
sampidx (int): Index for sample if multi-sample VCF
min_dp (int): Minimum depth to call site
major (float): Allele fraction to make unambiguous call
minor (float): Allele fraction to make ambiguous call
keep_tmp (bool): Do not delete temporary directory
quiet (bool): Do not write output to console
logfile (file): Append console output to this file
debug (bool): Print commands but do not run
Returns:
"""
# Check inputs
if vcf is None:
raise sysutils.PipelineStepError('VCF file is required')
# Outputs
out_fasta = os.path.join(outdir, 'consensus.fna')
sysutils.log_message('[--- vcf_to_consensus ---]\n', quiet, logfile)
sysutils.log_message('VCF: %s\n' % vcf, quiet, logfile)
# Parse VCF
chroms = []
samples = []
if os.path.splitext(vcf)[1] == '.gz':
lines = (l.decode('utf-8').strip('\n') for l in gzip.open(vcf, 'rb'))
else:
lines = (l.strip('\n') for l in open(vcf, 'r'))
# Parse headers
for l in lines:
if l.startswith('##'):
m = re.match('##contig=<ID=(\S+),length=(\d+)>', l)
if m:
chroms.append((m.group(1), int(m.group(2))))
else:
assert l.startswith('#')
cols = l.strip('#').split('\t')
samples = cols[9:]
break
if len(samples) <= sampidx:
msg = 'Sample index %d does not exist. Samples: %s' % (sampidx,
str(samples))
raise sysutils.PipelineStepError(msg)
chrom_ordered = [_[0] for _ in chroms]
chroms = dict(chroms)
newseqs = dict((c, ['.'] * chroms[c]) for c in list(chroms.keys()))
imputed = dict((c, [''] * chroms[c]) for c in list(chroms.keys()))
for l in lines:
chrom, start, stop, RA, AA, info, svals = parse_vcf_sample(l, sampidx)
gt = call_gt(RA, AA, svals, min_dp, major, minor)
if gt is None:
imputed[chrom][start - 1] = RA[0].lower()
else:
if len(gt) == 1:
newseqs[chrom][start - 1] = gt[0]
imputed[chrom][start - 1] = gt[0]
else:
if all(len(_) == 1 for _ in gt):
newseqs[chrom][start - 1] = sequtils.get_ambig(gt)
imputed[chrom][start - 1] = sequtils.get_ambig(gt)
else:
newseqs[chrom][start - 1] = ''.join(gt[0])
imputed[chrom][start - 1] = ''.join(gt[0])
# newseqs = imputed
sysutils.log_message('Output FASTA: %s\n' % out_fasta, quiet, logfile)
with open(out_fasta, 'w') as outh:
for chrom in chrom_ordered:
new_seqid = sequtils.update_seq_id(chrom, samples[sampidx])
new_seq = ''.join(newseqs[chrom]).replace('.', 'n')
m = re.match('^(?P<pre>n*)(?P<seq>[^n].+[^n])?(?P<suf>n*)$',
new_seq)
if m.group('seq') is None:
msg = u'%s\tFAIL\t%d\t%s\n' % (new_seqid, 0, u"👎🏼")
# Don't output sequence if not present
else:
msg = u'%s\tPASS\t%d\t%s\n' % (new_seqid, len(
m.group('seq')), u"👍🏼")
print('>%s SM:%s' % (new_seqid, samples[sampidx]), file=outh)
print(sequtils.wrap(new_seq), file=outh)
sysutils.log_message(msg, quiet, logfile)
return out_fasta
def ph_parser(
haplotypes_fa=None,
outdir='.',
prefix=None,
keep_gaps=False,
quiet=False,
logfile=None,
):
"""
Args:
haplotypes_fa (str): Path to haplotype file from PredictHaplo (fasta-ish)
outdir (str): Path to output directory
prefix (str): Prefix to add to sequence names
keep_gaps (bool): Do not remove gaps from alignment
quiet (bool): Do not write output to console
logfile (file): Append console output to this file
Returns:
"""
summary_txt = open(os.path.join(outdir, 'ph_summary.txt'), 'w')
newseq_fa = open(os.path.join(outdir, 'ph_haplotypes.fna'), 'w')
num_hap = 0
freq = []
fasta = []
newseq = None
ph = os.path.basename(haplotypes_fa).split(".")[0]
for l in open(haplotypes_fa, 'r'):
l = l.strip('\n')
if l.startswith('>'):
num_hap += 1
if newseq is not None:
fasta.append(newseq)
newseq = [ph, l.strip(">"), None, ""]
elif l.startswith(';'):
parts = l.strip(';').split(':')
if parts[0] == 'Freq':
freq.append(float(parts[1]))
newseq[2] = float(parts[1])
else:
pass
else:
newseq[3] += l.strip('\n')
fasta.append(newseq)
if len(fasta) == num_hap:
sysutils.log_message("Number of haplotypes is correct.\n", quiet,
logfile)
freq_sqrd = [x**2 for x in freq]
freq_sqrd_sum = sum(freq_sqrd)
hap_div = ((old_div(7000, (7000 - 1))) * (1 - freq_sqrd_sum))
print("PH_num_hap %s" % num_hap, file=summary_txt)
print("PH_hap_diversity %s" % hap_div, file=summary_txt)
seqlen = len(fasta[0][-1])
equal_len = True
for seq in fasta:
sl = len(seq[-1])
if sl != seqlen:
sysutils.log_message(
"Sequence length is different for each haplotype.\n",
quiet, logfile)
equal_len = False
else:
pass
if equal_len == True:
print("PH_seq_len %s" % seqlen, file=summary_txt)
for sub_list in fasta:
if prefix is None:
print('>sid|%s_%s|reg|%s| Freq=%s' %
(sub_list[0], sub_list[1], sub_list[0].split("_")[-1],
sub_list[2]),
file=newseq_fa)
else:
print('>sid|%s_%s_%s|reg|%s| Freq=%s' %
(prefix, sub_list[0], sub_list[1],
sub_list[0].split("_")[-1], sub_list[2]),
file=newseq_fa)
if keep_gaps:
print("%s" % (sub_list[-1]), file=newseq_fa)
else:
print("%s" % (sub_list[-1].replace('-', "")), file=newseq_fa)
sysutils.log_message(
"Summary and FASTA file completed for %s.\n" % haplotypes_fa,
quiet, logfile)
summary_txt.close()
newseq_fa.close()
def sample_reads(
fq1=None,
fq2=None,
fqU=None,
outdir='.',
nreads=None,
frac=None,
seed=None,
quiet=False,
logfile=None,
debug=False,
):
"""
Args:
fq1 (str): Path to fastq file with read 1
fq2 (str): Path to fastq file with read 2
fqU (str): Path to fastq file with unpaired reads
outdir (str): Path to output directory
nreads (int): Number of reads to sample
frac (float): Fraction of reads to sample
seed (int): Seed for random number generator
quiet (bool): Do not write output to console
logfile (file): Append console output to this file
debug (bool): Print commands but do not run
Returns:
out1 (str): Path to sampled fastq file with read 1
out2 (str): Path to sampled fastq file with read 2
outU (str): Path to sampled fastq file with unpaired reads
"""
# Check inputs
if fq1 is not None and fq2 is not None and fqU is None:
input_reads = "paired" # Paired end
elif fq1 is None and fq2 is None and fqU is not None:
input_reads = "single" # Single end
elif fq1 is not None and fq2 is not None and fqU is not None:
input_reads = "both"
else:
msg = "incorrect input reads; requires either "
msg += "(--fq1 AND --fq2) OR (--fqU) OR (--fq1 AND --fq2 AND --fqU)"
raise MissingRequiredArgument(msg)
# Check dependencies
sysutils.check_dependency('seqtk')
# Set seed
seed = seed if seed is not None else random.randrange(1, 1000)
sysutils.log_message('[--- sample_reads ---] Random seed = %d\n' % seed,
quiet, logfile)
# Set nreads/frac
if frac is not None:
if frac <= 0 or frac > 1:
raise sysutils.PipelineStepError('--frac must be > 0 and <= 1.')
frac_arg = '%f' % frac
else:
frac_arg = '%d' % nreads
cmds = None
if input_reads == 'single':
out1 = out2 = None
outU = os.path.join(outdir, 'sample_U.fastq')
cmds = [
[
'seqtk',
'sample',
'-s%d' % seed,
fqU,
frac_arg,
'>',
outU,
],
]
elif input_reads == 'paired':
out1 = os.path.join(outdir, 'sample_1.fastq')
out2 = os.path.join(outdir, 'sample_2.fastq')
outU = None
cmds = [
[
'seqtk',
'sample',
'-s%d' % seed,
fq1,
frac_arg,
'>',
out1,
],
[
'seqtk',
'sample',
'-s%d' % seed,
fq2,
frac_arg,
'>',
out2,
],
]
elif input_reads == 'both':
out1 = os.path.join(outdir, 'sample_1.fastq')
out2 = os.path.join(outdir, 'sample_2.fastq')
outU = os.path.join(outdir, 'sample_U.fastq')
cmds = [
[
'seqtk',
'sample',
'-s%d' % seed,
fq1,
frac_arg,
'>',
out1,
],
[
'seqtk',
'sample',
'-s%d' % seed,
fq2,
frac_arg,
'>',
out2,
],
[
'seqtk',
'sample',
'-s%d' % seed,
fqU,
frac_arg,
'>',
outU,
],
]
sysutils.command_runner(cmds, 'sample_reads', quiet, logfile, debug)
return out1, out2, outU
def assemble_to_ref(qry_fa,
ref_fa,
outdir,
pad_fh=None,
quiet=False,
logfile=None,
debug=False):
"""
Args:
qry_fa:
ref_fa:
outdir:
pad_fh:
quiet:
logfile:
debug:
Returns:
"""
# Align query to reference
fil, til = align_nucmer(qry_fa,
ref_fa,
outdir,
quiet=quiet,
logfile=logfile,
debug=debug)
if debug:
return None
# Parse tiling rows
tr_byref = defaultdict(list)
for l in open(til, 'rU'):
tr = TilingRow(l)
tr_byref[tr.ref].append(tr)
# Load reference(s)
refs = sorted(tr_byref.keys())
ref_dict = {s.id: s for s in SeqIO.parse(ref_fa, 'fasta')}
sysutils.log_message('\nReferences: %s\n' % ', '.join(refs), quiet,
logfile)
scaffolds = {}
for ref in refs:
if pad_fh is not None:
empty = EmptyReferenceAlignment(str(ref_dict[ref].seq).lower())
print('%s%s' % (ref.ljust(40), empty.rseq().upper()), file=pad_fh)
scaffolds[ref] = EmptyReferenceAlignment(
str(ref_dict[ref].seq).lower())
# Rank hits so that worst hit is in index 0 (best at the end)
ranked = sorted(tr_byref[ref], key=lambda x: x.pid)
ranked.sort(key=lambda x: x.qry_alen)
for tr in ranked:
out = show_aligns(tr.ref, tr.qry, fil)
out = out.decode()
# May be multiple alignments
flag = False
aln_reports = []
for l in out.strip('\n').split('\n'):
if re.match('^--\s+BEGIN', l):
aln_reports.append(list())
flag = True
if flag:
aln_reports[-1].append(l)
if re.match('^--\s+END', l):
flag = False
for aln_report in aln_reports:
# if debug:
# print("*" * 80, file=sys.stderr)
# print('\n'.join(aln_report), file=sys.stderr)
# print("*" * 80, file=sys.stderr)
nucaln = NucmerReferenceAlignment(aln_report)
# print('%d-%d' % (nucaln.rstart, nucaln.rend))
if pad_fh is not None:
pad = empty.merge_alignments(nucaln)
print('%s%s' % (tr.qry.ljust(40), pad.padded()),
file=pad_fh)
scaffolds[ref] = scaffolds[ref].merge_alignments(nucaln)
return scaffolds
def cliquesnv(fq1=None,
fq2=None,
fqU=None,
ref_fa=None,
outdir='.',
jardir='.',
O22min=None,
O22minfreq=None,
printlog=None,
single=False,
merging=None,
fasta_format='extended4',
outputstart=None,
outputend=None,
keep_tmp=False,
quiet=False,
logfile=None,
debug=False,
ncpu=1):
# check if paired vs. single
if fq1 is None and fq2 is None and fqU is not None:
single = True
# check dependencies and required arguments
if fq1 is None and fq2 is None and fqU is None:
raise MissingRequiredArgument("No fastq files given.")
if single == False and (fq1 is None or fq2 is None):
raise MissingRequiredArgument("Either fq1 or fq2 missing.")
if ref_fa is None:
raise MissingRequiredArgument("Reference FASTA missing.")
sysutils.check_dependency('samtools')
sysutils.check_dependency('bwa')
if (os.path.isfile(os.path.join(jardir, "clique-snv.jar"))):
print("CliqueSNV JAR file found.")
else:
raise MissingRequiredArgument("No JAR file found.")
# Temporary directory
tempdir = sysutils.create_tempdir('clique_snv', None, quiet, logfile)
# Load reference fasta
refs = {s.id: s for s in SeqIO.parse(ref_fa, 'fasta')}
# Identify reconstruction regions
regions = []
for rname, s in refs.items():
regions.append(('cs%02d' % (len(regions) + 1), rname, 1, len(s)))
sysutils.log_message('[--- Haplotype Reconstruction Regions ---]\n', quiet,
logfile)
for iv in regions:
sysutils.log_message('%s -- %s:%d-%d\n' % iv, quiet, logfile)
if single == False: #paired end
# remove .1 and .2 from read names
fq1_c = os.path.join(tempdir, "fq1_corrected.fastq")
fq2_c = os.path.join(tempdir, "fq2_corrected.fastq")
cmd01 = ["cat %s | sed 's/\.1 / /' > %s" % (fq1, fq1_c)]
cmd02 = ["cat %s | sed 's/\.2 / /' > %s" % (fq2, fq2_c)]
sysutils.command_runner([cmd01, cmd02], 'clique_snv:setup', quiet,
logfile, debug)
# Create alignment for each REFERENCE in the reconstruction regions
alnmap = {}
for cs, rname, spos, epos in regions:
if rname not in alnmap:
# Create alignment
tmp_ref_fa = os.path.join(tempdir, 'ref.%d.fa' % len(alnmap))
tmp_sam = os.path.join(tempdir, 'aligned.%d.sam' % len(alnmap))
SeqIO.write(refs[rname], tmp_ref_fa, 'fasta')
cmd1 = [
'bwa',
'index',
tmp_ref_fa,
]
cmd2 = [
'bwa',
'mem',
tmp_ref_fa,
fq1_c,
fq2_c,
'|',
'samtools',
'view',
'-h',
'-F',
'12',
'>',
tmp_sam,
]
cmd3 = ['rm', '-f', '%s.*' % tmp_ref_fa]
sysutils.command_runner([cmd1, cmd2, cmd3], 'clique_snv:setup',
quiet, logfile, debug)
alnmap[rname] = (tmp_ref_fa, tmp_sam)
else: #single read
# Create alignment for each REFERENCE in the reconstruction regions
alnmap = {}
for cs, rname, spos, epos in regions:
if rname not in alnmap:
# Create alignment
tmp_ref_fa = os.path.join(tempdir, 'ref.%d.fa' % len(alnmap))
tmp_sam = os.path.join(tempdir, 'aligned.%d.sam' % len(alnmap))
SeqIO.write(refs[rname], tmp_ref_fa, 'fasta')
cmd1 = [
'bwa',
'index',
tmp_ref_fa,
]
cmd2 = [
'bwa',
'mem',
tmp_ref_fa,
fqU,
'|',
'samtools',
'view',
'-h',
'-F',
'12',
'>',
tmp_sam,
]
cmd3 = ['rm', '-f', '%s.*' % tmp_ref_fa]
sysutils.command_runner([cmd1, cmd2, cmd3], 'clique_snv:setup',
quiet, logfile, debug)
alnmap[rname] = (tmp_ref_fa, tmp_sam)
# Run CliqueSNV for each region
cmd4 = ['mkdir -p %s' % os.path.join(outdir, 'clique_snv')]
sysutils.command_runner([
cmd4,
],
stage='cliquesnv',
quiet=quiet,
logfile=logfile,
debug=debug)
i = 0 #index for filenames
for cs, rname, spos, epos in regions:
msg = "Reconstruction region %s:" % cs
msg += " %s:%d-%d\n" % (rname, spos, epos)
sysutils.log_message(msg, quiet, logfile)
# rename the cliquesnv number (cs##) to include region (now: cs##_reg)
cs = '%s_%s' % (cs, rname.split('|')[-2])
samfile = os.path.join(tempdir, 'aligned.%d.sam' % i)
method = 'snv-illumina'
cmd5 = [
'java -jar %s -m %s -in %s -threads %d -outDir %s -fdf %s' %
(os.path.join(jardir, 'clique-snv.jar'), method, samfile, ncpu,
tempdir, fasta_format)
]
if O22min is not None:
cmd5 += ['-t %f' % O22min]
if O22minfreq is not None:
cmd5 += ['-tf %f' % O22minfreq]
if printlog is not None:
cmd5 += ['-log']
if merging is not None:
cmd5 += ['-cm %s' % merging]
if outputstart is not None:
cmd5 += ['-os %d' % outputstart]
if outputend is not None:
cmd5 += ['-oe %d' % outputend]
sysutils.command_runner([
cmd5,
],
stage='clique_snv',
quiet=quiet,
logfile=logfile,
debug=debug)
# copy output file and delete tempdir
outname1 = 'aligned.%d.txt' % i
outname2 = 'aligned.%d.fasta' % i
os.makedirs(os.path.join(outdir, 'clique_snv/%s' % cs), exist_ok=True)
if os.path.exists(os.path.join(tempdir, '%s' % outname1)):
shutil.copy(
os.path.join(tempdir, '%s' % outname1),
os.path.join(outdir, 'clique_snv/%s/%s.txt' % (cs, cs)))
if os.path.exists(os.path.join(tempdir, '%s' % outname2)):
shutil.copy(
os.path.join(tempdir, '%s' % outname2),
os.path.join(outdir, 'clique_snv/%s/%s.fasta' % (cs, cs)))
# parse output file
with open(
os.path.join(outdir,
'clique_snv/%s/%s_summary.txt' % (cs, cs)),
'w') as sumfile, open(
os.path.join(outdir, 'clique_snv/%s/%s.txt' % (cs, cs)),
'r') as infile:
l = infile.readlines()
freqs = []
haps = []
tempnum = ''
for line in l:
if "SNV got" in line:
tempnum = line.split(' ')[2]
if "frequency" in line:
freqs += [float(line.split(' ')[2][:-2])]
if "haplotype=" in line:
haps += [line.split('=')[1][1:-2]]
sumfile.write('CliqueSNV_num_hap\t%s\n' % tempnum)
freq_sqrd = [x**2 for x in freqs]
freq_sqrd_sum = sum(freq_sqrd)
hap_div = ((old_div(7000, (7000 - 1))) * (1 - freq_sqrd_sum))
sumfile.write('CliqueSNV_hap_diversity\t%s\n' % hap_div)
sumfile.write('CliqueSNV_seq_len\t%s\n' % len(haps[0]))
with open(os.path.join(outdir, 'clique_snv/%s/%s.fasta' % (cs, cs)),
'r') as fastafile:
fastadata = fastafile.read().replace('aligned.%d' % i, rname)
with open(
os.path.join(outdir, 'clique_snv/%s/%s.fasta' % (cs, cs)),
'w') as newfastafile:
newfastafile.write(fastadata)
i += 1
if not keep_tmp:
sysutils.remove_tempdir(tempdir, 'clique_snv', quiet, logfile)
return
def progressive_refine_assembly(
fq1=None, fq2=None, fqU=None, ref_fa=None, outdir='.',
max_step=None, subsample=None, seed=None, sample_id='sampleXX',
ncpu=1, xmx=sysutils.get_java_heap_size(),
keep_tmp=False, quiet=False, logfile=None, debug=False,
):
# Outputs
out_refined = os.path.join(outdir, 'refined.fna')
out_bt2 = os.path.join(outdir, 'refined_bt2.out')
out_summary = os.path.join(outdir, 'refined_summary.out')
#--- Initialize
cur_asm = ref_fa
cur_alnrate = None
assemblies = [OrderedDict(), ]
for s in SeqIO.parse(cur_asm, 'fasta'):
assemblies[-1][s.id] = s
# Message log for summary
summary = [
['iteration', 'alnrate', 'diffs'] + ['diff:%s' % s for s in assemblies[0].keys()]
]
# Seed random number generator
random.seed(seed)
for i in range(1, max_step+1):
# Generate a refined assembly
tmp_refined, tmp_bt2 = refine_assembly_step(
fq1=fq1, fq2=fq2, fqU=fqU, ref_fa=cur_asm, outdir=outdir,
iteration=i, subsample=subsample, sample_id=sample_id,
ncpu=ncpu, xmx=xmx, keep_tmp=keep_tmp,
quiet=True, logfile=logfile, debug=debug
)
# Check whether alignments are different
diffs = OrderedDict()
new_seqs = OrderedDict((s.id, s) for s in SeqIO.parse(tmp_refined, 'fasta'))
for id1, seq1 in new_seqs.items():
poss0 = [k for k in assemblies[-1].keys() if sequtils.seqid_match(id1, k)]
if len(poss0) == 1:
seq0 = assemblies[-1][poss0[0]]
else:
raise PipelineStepError("Could not match sequence %s" % id1)
alns = pairwise2.align.globalms(seq1.seq, seq0.seq, 2, -1, -3, -1)
d = min(sum(nc != cc for nc, cc in zip(t[0], t[1])) for t in alns)
diffs[id1] = d
total_diffs = sum(diffs.values())
# Check new alignment rate
with open(tmp_bt2, 'rU') as fh:
bt2str = fh.read()
m = re.search('(\d+\.\d+)\% overall alignment rate', bt2str)
if m is None:
msg = "Alignment rate not found in bowtie2 output."
msg += "Output file contents:\n%s\n" % bt2str
msg += "Aborting."
raise PipelineStepError(msg)
else:
new_alnrate = float(m.group(1))
# Create messages for log
row = [str(i), '%.02f' % new_alnrate, '%d' % total_diffs, ]
for k0 in assemblies[0].keys():
poss1 = [k for k in diffs.keys() if sequtils.seqid_match(k, k0)]
if len(poss1) == 0:
row.append('FAIL')
elif len(poss1) == 1:
row.append(str(diffs[poss1[0]]))
else:
raise PipelineStepError("Multiple matches for %s" % k0)
######row += list(map(str, diffs.values()))
summary.append(row)
# Create messages for console
sysutils.log_message('\nRefinement result:\n', quiet, logfile)
sysutils.log_message('\tDifferences:\n', quiet, logfile)
for s,d in diffs.items():
sysutils.log_message('\t\t%s\t%d\n' % (s,d), quiet, logfile)
if total_diffs > 0:
msg = '\t%d differences found with previous\n' % total_diffs
else:
msg = '\tNo differences with previous\n'
sysutils.log_message(msg, quiet, logfile)
if cur_alnrate is None:
msg = '\tAlignment rate: %0.2f\n' % new_alnrate
elif new_alnrate > cur_alnrate:
msg = '\tAlignment rate has improved: '
msg += '%.02f > %.02f\n' % (new_alnrate, cur_alnrate)
else:
msg = '\tAlignment rate has not improved: '
msg += '%.02f <= %.02f\n' % (new_alnrate, cur_alnrate)
sysutils.log_message(msg, quiet, logfile)
# Decide whether to keep going
keep_going = True
if total_diffs == 0:
keep_going = False
sysutils.log_message('Stopping: no differences found\n', quiet, logfile)
# We should also quit if alignment rate does not improve
# However, subsampling reads can lead to changes in alignment rate
# that can be ignore. When subsampling is implemented the first
# boolean value should check whether subsampling is enabled
if subsample is None: # not subsampling
if cur_alnrate is not None and new_alnrate <= cur_alnrate:
keep_going = False
msg = 'Stopping: alignment rate did not improve\n'
sysutils.log_message(msg, quiet, logfile)
cur_asm = tmp_refined
cur_alnrate = new_alnrate
assemblies.append(new_seqs)
if not keep_going:
break
# Final outputs
shutil.copy(cur_asm, out_refined)
shutil.copy(tmp_bt2, out_bt2)
with open(out_summary, 'w') as outh:
print('\n'.join('\t'.join(r) for r in summary), file=outh)
return out_refined, out_bt2, out_summary
def predict_haplo(
fq1=None,
fq2=None,
ref_fa=None,
region_txt=None,
outdir='.',
min_readlength=36,
keep_tmp=False,
quiet=False,
logfile=None,
debug=False,
):
""" Pipeline step to assemble haplotypes
Args:
fq1 (str): Path to fastq file with read 1
fq2 (str): Path to fastq file with read 2
ref_fa (str): Path to reference fasta file
region_txt (str): Path to region file
outdir (str): Path to output directory
min_readlength (int): Minimum readlength passed to PredictHaplo
keep_tmp (bool): Do not delete temporary directory
quiet (bool): Do not write output to console
logfile (file): Append console output to this file
debug (bool): Print commands but do not run
Returns:
best_fa (list): Path to best haplotype files (FASTA)
"""
# Check dependencies
sysutils.check_dependency('PredictHaplo-Paired')
sysutils.check_dependency('bwa')
# Temporary directory
tempdir = sysutils.create_tempdir('predict_haplo', None, quiet, logfile)
# Load reference fasta
refs = {s.id: s for s in SeqIO.parse(ref_fa, 'fasta')}
# Identify reconstruction regions
regions = []
if region_txt:
sysutils.log_message('Found regions file.\n', quiet, logfile)
for l in open(region_txt, 'r'):
rname, spos, epos = sequtils.region_to_tuple(l.strip())
if rname not in refs:
raise PipelineStepError("ERROR: reference %s not valid" %
rname)
spos = 1 if spos is None else spos
epos = len(refs[rname]) if epos is None else epos
regions.append(('PH%02d' % (len(regions) + 1), rname, spos, epos))
else:
for rname, s in refs.items():
regions.append(('PH%02d' % (len(regions) + 1), rname, 1, len(s)))
sysutils.log_message('[--- Haplotype Reconstruction Regions ---]\n', quiet,
logfile)
for iv in regions:
sysutils.log_message('%s -- %s:%d-%d\n' % iv, quiet, logfile)
# Create alignment for each REFERENCE in the reconstruction regions
alnmap = {}
for ph, rname, spos, epos in regions:
if rname not in alnmap:
# Create alignment
tmp_ref_fa = os.path.join(tempdir, 'ref.%d.fa' % len(alnmap))
tmp_sam = os.path.join(tempdir, 'aligned.%d.sam' % len(alnmap))
SeqIO.write(refs[rname], tmp_ref_fa, 'fasta')
cmd1 = [
'bwa',
'index',
tmp_ref_fa,
]
cmd2 = [
'bwa',
'mem',
tmp_ref_fa,
fq1,
fq2,
'|',
'samtools',
'view',
'-h',
'-F',
'12',
'>',
tmp_sam,
]
cmd3 = ['rm', '-f', '%s.*' % tmp_ref_fa]
sysutils.command_runner([cmd1, cmd2, cmd3], 'predict_haplo:setup',
quiet, logfile, debug)
alnmap[rname] = (tmp_ref_fa, tmp_sam)
best_fa = []
# Run PredictHaplo for each REGION
for ph, rname, spos, epos in regions:
msg = "Reconstruction region %s:" % ph
msg += " %s:%d-%d\n" % (rname, spos, epos)
sysutils.log_message(msg, quiet, logfile)
# Construct params specific for region
reg_params = dict(DEFAULTS)
reg_params['min_readlength'] = min_readlength
reg_params['reconstruction_start'] = spos
reg_params['reconstruction_stop'] = epos
reg_params['prefix'] = '%s_out.' % ph
# Lookup reference and alignment filename
reg_params['ref_fasta'] = os.path.basename(alnmap[rname][0])
reg_params['alignment'] = os.path.basename(alnmap[rname][1])
# Create config file for region
config_file = '%s.config' % ph
with open(os.path.join(tempdir, config_file), 'w') as outh:
tmpconfig = config_template % reg_params
print(tmpconfig.replace('###', '%'), file=outh)
try:
# Run PredictHaplo
cmd1 = [
'cd',
tempdir,
]
cmd2 = [
'PredictHaplo-Paired', config_file, '&>',
'%s.log' % config_file
]
sysutils.command_runner([
cmd1,
cmd2,
], 'predict_haplo:%s' % ph, quiet, logfile, debug)
# Copy files
dest = os.path.join(outdir, ph)
if not os.path.exists(dest):
os.makedirs(dest)
shutil.copy(os.path.join(tempdir, '%s.config.log' % ph), dest)
for f in glob(os.path.join(tempdir, '%s_out*global*.fas' % ph)):
shutil.copy(f, dest)
for f in glob(os.path.join(tempdir, '%s_out*global*.html' % ph)):
shutil.copy(f, dest)
bf, bh = rename_best(dest, ph)
best_fa.append((ph, bf))
except PipelineStepError as e:
print(e, file=sys.stderr)
if e.returncode == 139:
print("PredictHaplo segfaulted", file=sys.stderr)
best_fa.append((ph, None))
if not keep_tmp:
sysutils.remove_tempdir(tempdir, 'predict_haplo', quiet, logfile)
return best_fa
