from singlecellmultiomics.modularDemultiplexer.baseDemultiplexMethods import ScatteredUmiBarcodeDemuxMethod, UmiBarcodeDemuxMethod, NonMultiplexable, IlluminaBaseDemultiplexer, phredToFastqHeaderSafeQualities
from singlecellmultiomics.modularDemultiplexer.demultiplexModules import CELSeq2_c8_u6
import re
from singlecellmultiomics.utils import reverse_complement
class DamID2(UmiBarcodeDemuxMethod):
def __init__(self, barcodeFileParser, random_primer_read=None,random_primer_length=None, **kwargs):
self.barcodeFileAlias = 'DamID2'
UmiBarcodeDemuxMethod.__init__(
self,
umiRead=0,
umiStart=0,
umiLength=3,
barcodeRead=0,
barcodeStart=3,
barcodeLength=10,
random_primer_read=random_primer_read,
random_primer_length=random_primer_length,
barcodeFileAlias=self.barcodeFileAlias,
barcodeFileParser=barcodeFileParser,
**kwargs)
self.shortName = 'DamID2'
self.longName = 'DamID, CB: 10bp UMI: 3bp'
self.autoDetectable = True
self.description = '48 barcodes, 3bp umi followed by 10bp barcode, the last two bases of the barcode are CA'
self.sequenceCapture[0] = slice(
self.umiLength + self.barcodeLength - 1,
None) # do map the first base of the barcode
def demultiplex(self, records, probe=False, **kwargs):
if probe and records[0].sequence[self.barcodeLength + self.umiLength-2:self.barcodeLength + self.umiLength] != 'CA':
raise NonMultiplexable
# add first 2 bases as ligation tag:
ligation_start = self.umiLength + self.barcodeLength -2
ligation_end = ligation_start + 2
ligation_sequence = records[0].sequence[ligation_start:ligation_end]
ligation_qualities = records[0].qual[ligation_start:ligation_end]
taggedRecords = UmiBarcodeDemuxMethod.demultiplex(
self, records, probe=probe, **kwargs)
for rec in taggedRecords:
rec.addTagByTag(
'lh',
ligation_sequence,
isPhred=False,
make_safe=False)
rec.addTagByTag(
'lq',
ligation_qualities,
isPhred=True,
make_safe=False)
rec.tags['dt'] = 'DamID'
return taggedRecords
class DamID2_NO_OVERHANG(UmiBarcodeDemuxMethod):
def __init__(self, barcodeFileParser, random_primer_read=None,random_primer_length=None, **kwargs):
self.barcodeFileAlias = 'DamID2_8bp'
UmiBarcodeDemuxMethod.__init__(
self,
umiRead=0,
umiStart=0,
umiLength=3,
barcodeRead=0,
barcodeStart=3,
barcodeLength=8,
random_primer_read=random_primer_read,
random_primer_length=random_primer_length,
barcodeFileAlias=self.barcodeFileAlias,
barcodeFileParser=barcodeFileParser,
**kwargs)
self.shortName = 'DamID2_8bp_noCA'
self.longName = 'DamID, CB: 8bp UMI: 3bp'
self.autoDetectable = True
self.description = '48 barcodes, 3bp umi followed by 8bp barcode, no CA overhang in barcodes'
self.sequenceCapture[0] = slice(
self.umiLength + self.barcodeLength - 1,
None) # do map the first base of the barcode
def demultiplex(self, records, probe=False, **kwargs):
#if probe and records[0].sequence[self.barcodeLength + self.umiLength-2:self.barcodeLength + self.umiLength] != 'CA':
# raise NonMultiplexable
# add first 2 bases as ligation tag:
ligation_start = self.umiLength + self.barcodeLength
ligation_end = ligation_start + 2
ligation_sequence = records[0].sequence[ligation_start:ligation_end]
ligation_qualities = records[0].qual[ligation_start:ligation_end]
taggedRecords = UmiBarcodeDemuxMethod.demultiplex(
self, records, probe=probe, **kwargs)
for rec in taggedRecords:
rec.addTagByTag(
'lh',
ligation_sequence,
isPhred=False,
make_safe=False)
rec.addTagByTag(
'lq',
ligation_qualities,
isPhred=True,
make_safe=False)
rec.tags['dt'] = 'DamID'
return taggedRecords
class DamID2_c8_u3_cs2(UmiBarcodeDemuxMethod):
def __init__(self, barcodeFileParser, random_primer_read=None, random_primer_length=None, **kwargs):
self.barcodeFileAlias = 'DamAndT'
UmiBarcodeDemuxMethod.__init__(
self,
umiRead=0,
umiStart=0,
umiLength=3,
barcodeRead=0,
barcodeStart=3,
barcodeLength=10,
random_primer_read=random_primer_read,
random_primer_length=random_primer_length,
barcodeFileAlias=self.barcodeFileAlias,
barcodeFileParser=barcodeFileParser,
**kwargs)
self.shortName = 'DamAndT'
self.longName = 'DamAndT, CB: 10bp UMI: 3bp'
self.autoDetectable = True
self.description = 'DamID2 and CS2 Transcriptome'
self.sequenceCapture[0] = slice(
self.umiLength + self.barcodeLength - 2,
None) # do map the first two bases of the barcode
self.transcriptome_demux = CELSeq2_c8_u6(barcodeFileParser=barcodeFileParser,**kwargs)
self.damid_demux = DamID2(barcodeFileParser=barcodeFileParser,**kwargs)
def demultiplex(self, records, probe=False, **kwargs):
# Check if the supplied reads are mate-pair:
if len(records) != 2:
raise NonMultiplexable('Not mate pair')
# Obtain the DAMID barcode and umi:
try:
damid_tagged_records = self.damid_demux.demultiplex(records, probe=probe, **kwargs)
except NonMultiplexable as e:
damid_tagged_records = None
# Obtain TX barcode and umi
try:
tx_tagged_records = self.transcriptome_demux.demultiplex(records, probe=probe, **kwargs)
# Prune the poly T off R1 start
r1 = tx_tagged_records[0]
pos = 0
for pos, base in enumerate(r1.sequence):
if base!='T':
break
r1.sequence = r1.sequence[pos:]
r1.qualities = r1.qualities[pos:]
except NonMultiplexable:
tx_tagged_records = None
# Check which of the demuxers yielded results
# damid_tagged_records
# damid_taggedRecords[0].tags.update(ud)
# damid_taggedRecords[1].tags.update(ud)
if tx_tagged_records is not None and damid_tagged_records is not None:
# Both types
tagged_records = damid_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'Ambiguous'
elif tx_tagged_records is not None:
tagged_records = tx_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'RNA'
elif damid_tagged_records is not None:
tagged_records = damid_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'DamID'
elif tx_tagged_records is None and damid_tagged_records is None:
raise NonMultiplexable('Not DamID or TX')
else:
raise ValueError('failure')
return tagged_records
class DamID2_SCA(ScatteredUmiBarcodeDemuxMethod):
def __init__(self,
barcodeFileParser,
random_primer_read=None,
random_primer_length=None,
first_umi_len = 3,
first_bc_len = 4,
second_umi_len = 3,
second_barcode_len = 4,
barcode_alias=None,
**kwargs):
if barcode_alias is None:
self.barcodeFileAlias = 'DamID2_scattered_8bp'
else:
self.barcodeFileAlias= barcode_alias
# eg 3-ATTG-3-GAAC[GA]
self.total_mi_len = first_umi_len+first_bc_len+second_umi_len+second_barcode_len
barcode_slices = (
( slice(first_umi_len,first_umi_len+first_bc_len),
slice(first_umi_len+first_bc_len+second_umi_len,first_umi_len+first_bc_len+second_umi_len+second_barcode_len )), ()
)
umi_slices = (
(slice(0,first_umi_len),
slice(first_umi_len+first_bc_len,first_umi_len+first_bc_len+second_umi_len)
),
()
)
capture_slices = (
slice(self.total_mi_len,None),
slice(None)
)
ScatteredUmiBarcodeDemuxMethod.__init__(
self,
barcode_slices = barcode_slices, # 2-len Tuple of List of slices where the cell barcode is present in the read (slice(), slice(), ...], [ slice())
umi_slices = umi_slices, # 2-len List of slices where the UMI is present in the read [slice(), slice(), ...], [slice()] #
capture_slices = capture_slices, # Slices which indicate what region of read 1 and read2 to store in the final read (required)
barcodeFileAlias=self.barcodeFileAlias,
barcodeFileParser=barcodeFileParser,
**kwargs)
self.shortName = 'DamID2_3u4b3u6b'
self.longName = 'DamID, 3bp UMI, 4bp CB, 3bp UMI, 6bp CB'
self.autoDetectable = True
self.description = 'DamID, starting with a 3bp UMI, 4bp CB, 3bp UMI, 6bp CB'
def demultiplex(self, records, probe=False, **kwargs):
#if probe and records[0].sequence[self.barcodeLength + self.umiLength-2:self.barcodeLength + self.umiLength] != 'CA':
# raise NonMultiplexable
# add first 2 bases as ligation tag:
ligation_start = self.total_mi_len
ligation_end = self.total_mi_len+2
ligation_sequence = records[0].sequence[ligation_start:ligation_end]
ligation_qualities = records[0].qual[ligation_start:ligation_end]
taggedRecords = ScatteredUmiBarcodeDemuxMethod.demultiplex(
self, records, probe=probe, **kwargs)
for rec in taggedRecords:
rec.addTagByTag(
'lh',
ligation_sequence,
isPhred=False,
make_safe=False)
rec.addTagByTag(
'lq',
ligation_qualities,
isPhred=True,
make_safe=False)
return taggedRecords
class DamID2andT_SCA6(DamID2_c8_u3_cs2):
def __init__(self, barcodeFileParser, random_primer_read=None, random_primer_length=None, **kwargs):
self.transcriptome_demux = DamID2_SCA(barcodeFileParser=barcodeFileParser,barcode_alias='CS2_scattered_8bp',
first_umi_len = 3,
first_bc_len = 4,
second_umi_len = 3,
second_barcode_len = 4,
**kwargs)
self.damid_demux = DamID2_SCA(barcodeFileParser=barcodeFileParser,
second_barcode_len=6,
barcode_alias='DamID2_scattered_10bp',
**kwargs)
self.autoDetectable = True
self.shortName = 'DamID2andT_3u4b3u6b'
self.longName = 'DamID and transcriptome, 3bp UMI, 4bp CB, 3bp UMI, 6bp CB'
self.autoDetectable = True
self.description = 'DamID and transcriptome, starting with a 3bp UMI, 4bp CB, 3bp UMI, 6bp CB'
self.indexSummary = self.transcriptome_demux.indexSummary + ', ' + self.damid_demux.indexSummary
self.barcodeSummary = self.transcriptome_demux.barcodeSummary + ', ' + self.damid_demux.barcodeSummary
def demultiplex(self, records, probe=False, **kwargs):
# Check if the supplied reads are mate-pair:
if len(records) != 2:
raise NonMultiplexable('Not mate pair')
# Obtain the DAMID barcode and umi:
try:
damid_tagged_records = self.damid_demux.demultiplex(records, probe=probe, **kwargs)
except NonMultiplexable as e:
damid_tagged_records = None
# Obtain TX barcode and umi
try:
tx_tagged_records = self.transcriptome_demux.demultiplex(records, probe=probe, **kwargs)
# Prune the poly T off R1 start
r1 = tx_tagged_records[0]
pos = 0
for pos, base in enumerate(r1.sequence):
if base!='T':
break
r1.sequence = r1.sequence[pos:]
r1.qualities = r1.qualities[pos:]
except NonMultiplexable:
tx_tagged_records = None
# Check which of the demuxers yielded results
# damid_tagged_records
# damid_taggedRecords[0].tags.update(ud)
# damid_taggedRecords[1].tags.update(ud)
if tx_tagged_records is not None and damid_tagged_records is not None:
# Both types
for tx_tagged_record, damid_tagged_record in zip(tx_tagged_records, damid_tagged_records):
tx_tagged_record.tags.update(damid_tagged_record.tags)
tagged_records = tx_tagged_record
elif tx_tagged_records is not None:
tagged_records = tx_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'RNA'
elif damid_tagged_records is not None:
tagged_records = damid_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'DamID'
elif tx_tagged_records is None and damid_tagged_records is None:
raise NonMultiplexable('Not DamID or TX')
else:
raise ValueError('failure')
return tagged_records
class DamID2andT_SCA(DamID2_c8_u3_cs2):
def __init__(self, barcodeFileParser, random_primer_read=None, random_primer_length=None, **kwargs):
self.transcriptome_demux = DamID2_SCA(barcodeFileParser=barcodeFileParser,barcode_alias='CS2_scattered_8bp',
first_umi_len = 3,
first_bc_len = 4,
second_umi_len = 3,
second_barcode_len = 4,
**kwargs)
self.damid_demux = DamID2_SCA(barcodeFileParser=barcodeFileParser,
first_umi_len = 3,
first_bc_len = 4,
second_umi_len = 3,
second_barcode_len = 4,
barcode_alias='DamID2_scattered_8bp',
**kwargs
)
self.autoDetectable = True
self.shortName = 'DamID2andT_3u4b3u4b'
self.longName = 'DamID and transcriptome, 3bp UMI, 4bp CB, 3bp UMI, 4bp CB'
self.autoDetectable = True
self.description = 'DamID and transcriptome, starting with a 3bp UMI, 4bp CB, 3bp UMI, 4bp CB'
self.indexSummary = self.transcriptome_demux.indexSummary + ', ' + self.damid_demux.indexSummary
self.barcodeSummary = self.transcriptome_demux.barcodeSummary + ', ' + self.damid_demux.barcodeSummary
def demultiplex(self, records, probe=False, **kwargs):
# Check if the supplied reads are mate-pair:
if len(records) != 2:
raise NonMultiplexable('Not mate pair')
# Obtain the DAMID barcode and umi:
try:
damid_tagged_records = self.damid_demux.demultiplex(records, probe=probe, **kwargs)
except NonMultiplexable as e:
damid_tagged_records = None
# Obtain TX barcode and umi
try:
tx_tagged_records = self.transcriptome_demux.demultiplex(records, probe=probe, **kwargs)
# Prune the poly T off R1 start
r1 = tx_tagged_records[0]
pos = 0
for pos, base in enumerate(r1.sequence):
if base!='T':
break
r1.sequence = r1.sequence[pos:]
r1.qualities = r1.qualities[pos:]
except NonMultiplexable:
tx_tagged_records = None
# Check which of the demuxers yielded results
# damid_tagged_records
# damid_taggedRecords[0].tags.update(ud)
# damid_taggedRecords[1].tags.update(ud)
if tx_tagged_records is not None and damid_tagged_records is not None:
# Both types
tagged_records = damid_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'Ambiguous'
elif tx_tagged_records is not None:
tagged_records = tx_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'RNA'
elif damid_tagged_records is not None:
tagged_records = damid_tagged_records
for rec in tagged_records:
rec.tags['dt'] = 'DamID'
elif tx_tagged_records is None and damid_tagged_records is None:
raise NonMultiplexable('Not DamID or TX')
else:
raise ValueError('failure')
return tagged_records
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