def test_long_cigar_parsing_no_context(self):
cigar = "110M1I174M"
query = (
"AATCCAGCTAGCTGTGCAGCATCTGACAAGCTCTATTCAACTGCAGTAATGCTACCTCGTA"
"CTCACGCTTTCCAAGTGCTTGGCGTCGCATCTCGGTCCTTTGTACGCCGAAAAAATGGCCTG"
"ACAACTAAGCTACGGCACGCTGCCATGTTGGGTCATAACGATATCTCTGGTTCATCCGTGAC"
"CGAACATGTCATGGAGTAGCAGGAGCTATTAATTCGCGGAGGACAATGAGGTTCGTAGTCAC"
"TGTCTTCCGCAATCGTACATCGCTCCTGCAGGTGGCCT")
cutsite_window = 12
context = False
intBC, indels = alignment_utilities.parse_cigar(
cigar,
query,
self.long_ref,
0,
0,
self.long_barcode_interval,
self.long_cutsites,
cutsite_window,
context,
context_size=0,
)
self.assertEqual(intBC, "ATCTGACAAGCTCT")
expected_cuts = ["111:1I", "None", "None"]
for i in range(len(indels)):
self.assertEqual(indels[i], expected_cuts[i])
def test_basic_cigar_string_deletion(self):
cigar = "6M2D2M"
query = "ACGGTTTT"
cutsite_window = 1
context = False
intBC, indels = alignment_utilities.parse_cigar(
cigar,
query,
self.basic_ref,
0,
0,
self.basic_barcode_interval,
self.basic_cutsites,
cutsite_window,
context,
)
self.assertEqual(intBC, "GG")
self.assertEqual(len(self.basic_cutsites), len(indels))
self.assertEqual(indels[0], "7:2D")
def test_basic_cigar_string_match(self):
query = "ACGGTTAATT"
cigar = "10M"
cutsite_window = 1
context = False
intBC, indels = alignment_utilities.parse_cigar(
cigar,
query,
self.basic_ref,
0,
0,
self.basic_barcode_interval,
self.basic_cutsites,
cutsite_window,
context,
)
self.assertEqual(intBC, "GG")
self.assertEqual(len(self.basic_cutsites), len(indels))
self.assertEqual(indels[0], "None")
def test_basic_cigar_string_insertion_with_context(self):
cigar = "8M3I2M"
query = "ACGGTTAAGTGTT"
cutsite_window = 1
context = True
intBC, indels = alignment_utilities.parse_cigar(
cigar,
query,
self.basic_ref,
0,
0,
self.basic_barcode_interval,
self.basic_cutsites,
cutsite_window,
context,
context_size=1,
)
self.assertEqual(intBC, "GG")
self.assertEqual(len(self.basic_cutsites), len(indels))
self.assertEqual(indels[0], "A[9:3I]GTGT")
def test_complex_cigar_parsing_intersite_deletion(self):
cigar = "98M13D55M54D130M"
query = (
"AATCCAGCTAGCTGTGCAGCTGACAGGGAAGCAAATTCAACTGCAGTAATGCTACCTCGT"
"ACTCACGCTTTCCAAGTGCTTGGCGTCGCATCTCGGTCAAAAAGGCCTTACAACAAAGCTACTGA"
"ACGAAGCAATGTTAGGACATAAAGATATCGGAGGACAATGATGTAAGTAGTCACTGTCTTCCTAA"
"ATAGTCAAACTCTCCAGAACATTGAATAGAGGGCCCGAAAAAACCATCTCAAAAGCCTCTACACA"
"GACTTCTAGAATACAAACAACTGATCTT")
cutsite_window = 12
context = True
intBC, indels = alignment_utilities.parse_cigar(
cigar,
query,
self.long_ref,
0,
0,
self.long_barcode_interval,
self.long_cutsites,
cutsite_window,
context,
context_size=5,
)
self.assertEqual(intBC, "TGACAGGGAAGCAA")
expected_cuts = [
"CGGTC[99:13D]AAAAA",
"GATAT[167:54D]CGGAG",
"GATAT[167:54D]CGGAG",
]
for i in range(len(indels)):
self.assertEqual(indels[i], expected_cuts[i])
def test_intersite_deletion_parsing(self):
cigar = "112M108D173M"
query = (
"AATCCAGCTAGCTGTGCAGCTTGTTTTAAACCAGATTCAACTGCAGTAATGCTACCTCGT"
"ACTCACGCTTTCCAAGTGCTTGGCGTAGCATCTAGGTCCTAAGTACGCCGAACGGCTGACAATGC"
"GGTTCGTAGTCACTGTCTACCGCAAACGTCAATCGCTCATCCAGGTGGCCAAGAGGGCACGTTTA"
"CACACGCTGATCATCCTCGACTGTGCCCTCTAGTAGCCAGCCAGAGGTTGTGTGCCCCTCCCCCG"
"GGCCGTCCGTGACCCTGGAAGGTGCCACTC")
cutsite_window = 12
context = True
intBC, indels = alignment_utilities.parse_cigar(
cigar,
query,
self.long_ref,
0,
0,
self.long_barcode_interval,
self.long_cutsites,
cutsite_window,
context,
context_size=5,
)
self.assertEqual(intBC, "TTGTTTTAAACCAG")
expected_cuts = [
"CCGAA[113:108D]CGGCT",
"CCGAA[113:108D]CGGCT",
"CCGAA[113:108D]CGGCT",
]
for i in range(len(indels)):
self.assertEqual(indels[i], expected_cuts[i])
def call_alleles(
alignments: pd.DataFrame,
ref_filepath: Optional[str] = None,
ref: Optional[str] = None,
barcode_interval: Tuple[int, int] = (20, 34),
cutsite_locations: List[int] = [112, 166, 220],
cutsite_width: int = 12,
context: bool = True,
context_size: int = 5,
) -> pd.DataFrame:
"""Call indels from CIGAR strings.
Given many alignments, we extract the indels by comparing the CIGAR strings
of each alignment to the reference sequence.
Args:
alignments: Alignments provided in DataFrame
ref_filepath: Filepath to the reference sequence
ref: Nucleotide sequence of the reference
barcode_interval: Interval in reference corresponding to the integration
barcode
cutsite_locations: A list of all cutsite positions in the reference
cutsite_width: Number of nucleotides left and right of cutsite location
that indels can appear in.
context: Include sequence context around indels
context_size: Number of bases to the right and left to include as
context
Returns:
A DataFrame mapping each sequence alignment to the called indels.
"""
if (ref is None) == (ref_filepath is None):
raise PreprocessError(
"Either `ref_filepath` or `ref` must be provided.")
alignment_to_indel = {}
alignment_to_intBC = {}
if ref_filepath:
ref = str(list(SeqIO.parse(ref_filepath, "fasta"))[0].seq)
for _, row in tqdm(
alignments.iterrows(),
total=alignments.shape[0],
desc="Parsing CIGAR strings into indels",
):
intBC, indels = alignment_utilities.parse_cigar(
row.CIGAR,
row.Seq,
ref,
row.ReferenceBegin,
row.QueryBegin,
barcode_interval,
cutsite_locations,
cutsite_width,
context=context,
context_size=context_size,
)
alignment_to_indel[row.readName] = indels
alignment_to_intBC[row.readName] = intBC
indel_df = pd.DataFrame.from_dict(
alignment_to_indel,
orient="index",
columns=[f"r{i}" for i in range(1,
len(cutsite_locations) + 1)],
)
indel_df["allele"] = indel_df.apply(
lambda x: "".join([str(i) for i in x.values]), axis=1)
indel_df["intBC"] = indel_df.index.map(alignment_to_intBC)
alignments.set_index("readName", inplace=True)
alignments = alignments.join(indel_df)
alignments.reset_index(inplace=True)
# check cut-sites and raise a warning if any missing data is detected
cutsites = utilities.get_default_cut_site_columns(alignments)
if np.any((alignments[cutsites] == "").sum(axis=0) > 0):
warnings.warn(
"Detected missing data in alleles. You might"
" consider re-running align_sequences with a"
" lower gap-open penalty, or using a separate"
" alignment strategy.",
PreprocessWarning,
)
return alignments