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Anchor barcodes during demultiplexing #58

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merged 17 commits into from
Dec 5, 2023
Merged

Anchor barcodes during demultiplexing #58

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97fe0c1
Ease the process of anchoring the barcodes at the 5' sites
vaamb Nov 24, 2023
eb34dd4
Use `-g` instead of `--front` to be more consistent with the reverse`-G`
vaamb Nov 24, 2023
088ce17
Add `TestDemuxSingle.test_anchored`
vaamb Nov 24, 2023
f726858
Add `TestDemuxSingle.test_anchored_cut`
vaamb Nov 24, 2023
aafaa08
Add `TestDemuxPaired.test_anchored`
vaamb Nov 24, 2023
09c7817
Add checks to be sure both barcodes are anchored when using `demux_pa…
vaamb Nov 24, 2023
04eeaf6
Add `TestDemuxPaired.test_mixed_orientation_anchored`
vaamb Nov 24, 2023
829a387
Fix lint issues
vaamb Nov 24, 2023
4b8014b
Apply suggestions from code review
vaamb Dec 1, 2023
40c1963
Fix linting issues
vaamb Dec 1, 2023
b21237e
Raise an error for when trying to anchor the reverse barcode without …
vaamb Dec 1, 2023
383d7ff
Add `TestDemuxPaired.test_dual_index_anchored`
vaamb Dec 5, 2023
ed4136a
Nits
vaamb Dec 5, 2023
3217f5f
Add `_check_paired_requirements` helper function
vaamb Dec 5, 2023
1fe52d8
Remove pylint comment
vaamb Dec 5, 2023
29ba89e
Fix linting issue
vaamb Dec 5, 2023
9b04a7b
whitespace
colinvwood Dec 5, 2023
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81 changes: 58 additions & 23 deletions q2_cutadapt/_demux.py
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Original file line number Diff line number Diff line change
Expand Up @@ -40,9 +40,12 @@ def run_command(cmd, verbose=True):

def _build_demux_command(seqs_dir_fmt, barcode_fhs, per_sample_dir_fmt,
untrimmed_dir_fmt, error_rate, minimum_length,
forward_cut=0, reverse_cut=0, cores=1):
forward_cut=0, reverse_cut=0,
anchor_forward=False, anchor_reverse=False,
cores=1):
cmd = ['cutadapt',
'--front', 'file:%s' % barcode_fhs['fwd'].name,
'-g',
f'{"^" if anchor_forward else ""}file:{barcode_fhs["fwd"].name}',
'--error-rate', str(error_rate),
'--minimum-length', str(minimum_length),
# {name} is a cutadapt convention for interpolating the sample id
Expand All @@ -57,7 +60,8 @@ def _build_demux_command(seqs_dir_fmt, barcode_fhs, per_sample_dir_fmt,
# Dual indices
cmd += [
'--pair-adapters',
'-G', 'file:%s' % barcode_fhs['rev'].name,
'-G',
f'{"^" if anchor_reverse else ""}file:{barcode_fhs["rev"].name}', # noqa: E501
]
cmd += [
'-p', os.path.join(str(per_sample_dir_fmt), '{name}.2.fastq.gz'),
Expand All @@ -66,7 +70,7 @@ def _build_demux_command(seqs_dir_fmt, barcode_fhs, per_sample_dir_fmt,
str(seqs_dir_fmt.forward_sequences.view(FastqGzFormat)),
str(seqs_dir_fmt.reverse_sequences.view(FastqGzFormat)),
'-U', str(reverse_cut),
]
]
else:
# SINGLE-END
cmd += [str(seqs_dir_fmt.file.view(FastqGzFormat))]
Expand Down Expand Up @@ -196,7 +200,7 @@ def _check_barcodes_uniqueness(

def _demux(seqs, per_sample_sequences, forward_barcodes, reverse_barcodes,
error_tolerance, mux_fmt, batch_size, minimum_length, forward_cut,
reverse_cut, cores):
reverse_cut, anchor_forward, anchor_reverse, cores):
fwd_barcode_name = forward_barcodes.name
forward_barcodes = forward_barcodes.drop_missing_values()
barcodes = forward_barcodes.to_series().to_frame()
Expand All @@ -222,11 +226,11 @@ def _demux(seqs, per_sample_sequences, forward_barcodes, reverse_barcodes,
open_fhs['rev'] = tempfile.NamedTemporaryFile()
_write_barcode_fasta(barcode_batch[rev_barcode_name],
open_fhs['rev'])
cmd = _build_demux_command(previous_untrimmed, open_fhs,
per_sample_sequences,
current_untrimmed, error_tolerance,
minimum_length, forward_cut, reverse_cut,
cores)
cmd = _build_demux_command(
previous_untrimmed, open_fhs, per_sample_sequences,
current_untrimmed, error_tolerance, minimum_length, forward_cut,
reverse_cut, anchor_forward, anchor_reverse, cores
)
run_command(cmd)
open_fhs['fwd'].close()
if reverse_barcodes is not None:
Expand All @@ -243,10 +247,11 @@ def _demux(seqs, per_sample_sequences, forward_barcodes, reverse_barcodes,

def demux_single(seqs: MultiplexedSingleEndBarcodeInSequenceDirFmt,
barcodes: qiime2.CategoricalMetadataColumn,
cut: int = 0,
anchor_barcode: bool = False,
error_rate: float = 0.1,
batch_size: int = 0,
minimum_length: int = 1,
cut: int = 0,
cores: int = 1) -> \
(CasavaOneEightSingleLanePerSampleDirFmt,
MultiplexedSingleEndBarcodeInSequenceDirFmt):
Expand All @@ -255,17 +260,53 @@ def demux_single(seqs: MultiplexedSingleEndBarcodeInSequenceDirFmt,
mux_fmt = MultiplexedSingleEndBarcodeInSequenceDirFmt

untrimmed = _demux(
seqs, per_sample_sequences, barcodes, None, error_rate, mux_fmt,
batch_size, minimum_length, cut, 0, cores)
seqs, per_sample_sequences, barcodes, None, error_rate,
mux_fmt, batch_size, minimum_length, cut, 0, anchor_barcode, False,
cores)

return per_sample_sequences, untrimmed


def _check_paired_requirements(loc):
mixed_orientation = loc.get("mixed_orientation", None)
forward_cut = loc.get("forward_cut", 0)
reverse_cut = loc.get("reverse_cut", 0)
reverse_barcodes = loc.get("reverse_barcodes", None)
anchor_forward_barcode = loc.get("anchor_forward_barcode", False)
anchor_reverse_barcode = loc.get("anchor_reverse_barcode", False)

if (
not mixed_orientation
and anchor_reverse_barcode and (reverse_barcodes is None)
):
raise ValueError("A reverse barcode needs to be provided in order to "
"anchor the reverse barcode.")

if (
mixed_orientation
and forward_cut != reverse_cut
):
raise ValueError("'forward_cut' and 'reverse_cut' need to be set to "
"the same number when using the 'mixed_orientation' "
"mode.")

if (
mixed_orientation
and anchor_forward_barcode != anchor_reverse_barcode
):
colinvwood marked this conversation as resolved.
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raise ValueError(
"'anchor_forward_barcode' and 'anchor_reverse_barcode' need to be "
"set to the same value when using the 'mixed_orientation' mode."
)


def demux_paired(seqs: MultiplexedPairedEndBarcodeInSequenceDirFmt,
forward_barcodes: qiime2.CategoricalMetadataColumn,
reverse_barcodes: qiime2.CategoricalMetadataColumn = None,
forward_cut: int = 0,
reverse_cut: int = 0,
anchor_forward_barcode: bool = False,
anchor_reverse_barcode: bool = False,
error_rate: float = 0.1,
batch_size: int = 0,
minimum_length: int = 1,
Expand All @@ -275,22 +316,15 @@ def demux_paired(seqs: MultiplexedPairedEndBarcodeInSequenceDirFmt,
MultiplexedPairedEndBarcodeInSequenceDirFmt):
_check_barcodes_uniqueness(
forward_barcodes, reverse_barcodes, mixed_orientation)

if (
mixed_orientation
and forward_cut != reverse_cut
):
raise ValueError("'forward_cut' and 'reverse_cut' need to be set to "
"the same number when using the 'mixed_orientation' "
"mode")
_check_paired_requirements(locals())

per_sample_sequences = CasavaOneEightSingleLanePerSampleDirFmt()
mux_fmt = MultiplexedPairedEndBarcodeInSequenceDirFmt

untrimmed = _demux(
seqs, per_sample_sequences, forward_barcodes, reverse_barcodes,
error_rate, mux_fmt, batch_size, minimum_length, forward_cut,
reverse_cut, cores)
reverse_cut, anchor_forward_barcode, anchor_reverse_barcode, cores)

if mixed_orientation:
fwd = untrimmed.forward_sequences.view(FastqGzFormat)
Expand All @@ -305,6 +339,7 @@ def demux_paired(seqs: MultiplexedPairedEndBarcodeInSequenceDirFmt,
untrimmed = _demux(
remaining_seqs, per_sample_sequences, forward_barcodes,
reverse_barcodes, error_rate, mux_fmt, batch_size,
minimum_length, 0, 0, cores)
minimum_length, 0, 0, anchor_reverse_barcode,
anchor_forward_barcode, cores)

return per_sample_sequences, untrimmed
25 changes: 21 additions & 4 deletions q2_cutadapt/plugin_setup.py
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Original file line number Diff line number Diff line change
Expand Up @@ -263,6 +263,7 @@
'barcodes': MetadataColumn[Categorical],
'error_rate': Float % Range(0, 1, inclusive_start=True,
inclusive_end=True),
'anchor_barcode': Bool,
'batch_size': Int % Range(0, None),
'minimum_length': Int % Range(1, None),
'cut': Int,
Expand All @@ -283,6 +284,10 @@
'allowable error rate. The default value specified by '
'cutadapt is 0.1 (=10%), which is greater than '
'`demux emp-*`, which is 0.0 (=0%).',
'anchor_barcode': 'Anchor the barcode. The barcode is then '
'expected to occur in full length at the beginning '
'(5\' end) of the sequence. Can speed up '
'demultiplexing if used.',
'batch_size': 'The number of samples cutadapt demultiplexes '
'concurrently. Demultiplexing in smaller batches will '
'yield the same result with marginal speed loss, and '
Expand All @@ -292,11 +297,11 @@
'the cutadapt default of 0 has been overridden, '
'because that value produces empty sequence '
'records.',
'cut': 'Remove the specified number of bases from the sequences. Bases'
'are removed before demultiplexing. If a positive value is'
'provided, bases are removed from the beginning of the '
'cut': 'Remove the specified number of bases from the sequences. '
'Bases are removed before demultiplexing. If a positive value '
'is provided, bases are removed from the beginning of the '
'sequences. If a negative value is provided, bases are removed '
'from the end of the sequences',
'from the end of the sequences.',
},
output_descriptions={
'per_sample_sequences': 'The resulting demultiplexed sequences.',
Expand Down Expand Up @@ -324,6 +329,8 @@
'reverse_cut': Int,
'error_rate': Float % Range(0, 1, inclusive_start=True,
inclusive_end=True),
'anchor_forward_barcode': Bool,
'anchor_reverse_barcode': Bool,
'batch_size': Int % Range(0, None),
'minimum_length': Int % Range(1, None),
'mixed_orientation': Bool,
Expand Down Expand Up @@ -358,6 +365,16 @@
'the sequences. If --p-mixed-orientation is set, then '
'both --p-forward-cut and --p-reverse-cut must be '
'set to the same value.',
'anchor_forward_barcode': 'Anchor the forward barcode. The '
'barcode is then expected to occur in full '
'length at the beginning (5\' end) of the '
'forward sequence. Can speed up '
'demultiplexing if used.',
'anchor_reverse_barcode': 'Anchor the reverse barcode. The '
'barcode is then expected to occur in full '
'length at the beginning (5\' end) of the '
'reverse sequence. Can speed up '
'demultiplexing if used.',
'error_rate': 'The level of error tolerance, specified as the maximum '
'allowable error rate.',
'batch_size': 'The number of samples cutadapt demultiplexes '
Expand Down
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