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from Bio import AlignIO, SeqIO
from Bio.Seq import Seq
from Bio.SeqIO import SeqRecord
from Bio.Align import MultipleSeqAlignment
import logging
from pathlib import Path
import re
from collections import namedtuple, defaultdict, Counter
import bisect
import numpy as np
from tqdm import tqdm
from parsnp.logger import logger, TqdmToLogger, MIN_TQDM_INTERVAL
import spoa
#%%
# MAX_LEN = 20
MIN_LEN = 10
BASE_LENGTH = 10
length_window = 0.10
window_prop = 0.75
IdPair = namedtuple('IdPair', 'gid cid')
Segment = namedtuple("Segment", "idp start stop strand")
def parse_xmfa_header(xmfa_file):
index_to_gid = {}
gid_to_index = {}
with open(xmfa_file) as parsnp_fd:
for line in (line.strip() for line in parsnp_fd):
if line[:2] == "##":
if line.startswith("##SequenceIndex"):
index = line.split(' ')[1]
elif line.startswith("##SequenceFile"):
gid = Path(line.split(' ')[1]).stem
elif line.startswith("##SequenceLength"):
gid_to_index[gid] = index
index_to_gid[index] = gid
elif line[0] != "#":
break
return index_to_gid, gid_to_index
def index_input_sequences(xmfa_file, file_list):
basename_to_path = {}
for f in file_list:
basename = str(Path(f).stem)
basename_to_path[basename] = f
gid_to_records = {}
gid_to_cid_to_index = {}
gid_to_index_to_cid = {}
with open(xmfa_file) as parsnp_fd:
for line in (line.strip() for line in parsnp_fd):
if line[:2] == "##":
if line.startswith("##SequenceFile"):
basename = Path(line.split(' ')[1]).stem
p = Path(basename_to_path[basename])
gid_to_records[p.stem] = {}
gid_to_cid_to_index[p.stem] = {}
gid_to_index_to_cid[p.stem] = {}
for idx, rec in enumerate(SeqIO.parse(str(p), "fasta")):
gid_to_records[p.stem][rec.id] = rec
gid_to_cid_to_index[p.stem][rec.id] = idx + 1
gid_to_index_to_cid[p.stem][idx + 1] = rec.id
return gid_to_records, gid_to_cid_to_index, gid_to_index_to_cid
def xmfa_to_covered(xmfa_file, index_to_gid, gid_to_index_to_cid):
seqid_parser = re.compile(r'^cluster(\d+) s(\d+):p(\d+)/.*')
idpair_to_segments = defaultdict(list)
cluster_to_named_segments = defaultdict(list)
for aln in AlignIO.parse(xmfa_file, "mauve"):
for seq in aln:
# Skip reference for now...
aln_len = seq.annotations["end"] - seq.annotations["start"]
cluster_idx, contig_idx, startpos = [int(x) for x in seqid_parser.match(seq.id).groups()]
gid = index_to_gid[seq.name]
if seq.annotations["strand"] == -1:
startpos, endpos = startpos - aln_len, startpos
else:
endpos = startpos + aln_len
idp = IdPair(gid, gid_to_index_to_cid[gid][contig_idx])
seg = Segment(idp, startpos, startpos + aln_len, seq.annotations["strand"])
idpair_to_segments[idp].append(seg)
cluster_to_named_segments[cluster_idx].append(seg)
for idp in idpair_to_segments:
idpair_to_segments[idp] = sorted(idpair_to_segments[idp])
return idpair_to_segments, cluster_to_named_segments
def run_msa(downstream_segs_to_align, gid_to_records):
keep_extending = True
iteration = 0
seq_lens = [seg.stop - seg.start for seg in downstream_segs_to_align]
longest_seq = max(seq_lens)
mean_seq_len = np.mean(seq_lens)
# if sum(
# mean_seq_len*(1 - length_window) <= (seg.stop - seg.start) <= mean_seq_len*(1 + length_window) for seg in downstream_segs_to_align) > len(downstream_segs_to_align)*window_prop:
# base_length = int(mean_seq_len*(1 + length_window))
# else:
# base_length = BASE_LENGTH
base_length = BASE_LENGTH
while keep_extending:
seqs_to_align = ["A" + (str(
gid_to_records[seg.idp.gid][seg.idp.cid].seq[seg.start:seg.stop] if seg.strand == 1
else gid_to_records[seg.idp.gid][seg.idp.cid].seq[seg.start:seg.stop].reverse_complement()
)[:base_length*(2**iteration)]) for seg in downstream_segs_to_align]
# print(seqs_to_align)
consensus, aligned_msa_seqs = spoa.poa(seqs_to_align)
consensus = consensus.replace("A", "", 1)
aligned_msa_seqs = [s.replace("A", "", 1) for s in aligned_msa_seqs]
indel_cols = 0
for i in range(len(aligned_msa_seqs[0])):
dash_frac = sum(msa_seq[i] == "-" for msa_seq in aligned_msa_seqs) / len(aligned_msa_seqs)
if dash_frac > 0.5:
indel_cols += 1
if indel_cols >= 10:
keep_extending = False
if i-indel_cols < MIN_LEN:
aligned_msa_seqs = ["" for msa_seq in aligned_msa_seqs]
else:
aligned_msa_seqs = [msa_seq[:i-indel_cols] for msa_seq in aligned_msa_seqs]
break
else:
indel_cols = 0
if BASE_LENGTH*(2**iteration) > longest_seq:
keep_extending = False
break
iteration += 1
return aligned_msa_seqs
def extend_clusters(xmfa_file, gid_to_index, gid_to_cid_to_index, idpair_to_segments, cluster_to_named_segments, gid_to_records):
ret_lcbs = []
seqid_parser = re.compile(r'^cluster(\d+) s(\d+):p(\d+)/.*')
for aln_idx, aln in enumerate(tqdm(
AlignIO.parse(xmfa_file, "mauve"),
total=len(cluster_to_named_segments),
file=TqdmToLogger(logger, level=logging.INFO),
mininterval=MIN_TQDM_INTERVAL)):
# validate_lcb(aln, gid_to_records, parsnp_header=True)
seq = aln[0]
cluster_idx, contig_idx, startpos = [int(x) for x in seqid_parser.match(seq.id).groups()]
segs = cluster_to_named_segments[cluster_idx]
# Get upstream sequences
assert(segs[0].strand == 1)
upstream_segs_to_align = []
downstream_segs_to_align = []
for covered_seg in segs:
seg_idx = bisect.bisect_left(idpair_to_segments[covered_seg.idp], covered_seg)
if covered_seg.strand == 1:
uncovered_start = covered_seg.stop
if seg_idx == len(idpair_to_segments[covered_seg.idp]) - 1:
uncovered_stop = len(gid_to_records[covered_seg.idp.gid][covered_seg.idp.cid].seq)
else:
uncovered_stop = idpair_to_segments[covered_seg.idp][seg_idx+1].start
else:
uncovered_stop = covered_seg.start
if seg_idx == 0:
uncovered_start = 0
else:
uncovered_start = idpair_to_segments[covered_seg.idp][seg_idx-1].stop
downstream_segs_to_align.append(Segment(covered_seg.idp, uncovered_start, uncovered_stop, covered_seg.strand))
aligned_msa_seqs = run_msa(downstream_segs_to_align, gid_to_records)
new_lcb = MultipleSeqAlignment([])
# Assumes alignments are always in the same order
new_bp = []
for seg_idx, (covered_seg, uncovered_seg, aln_str) in enumerate(zip(segs, downstream_segs_to_align, aligned_msa_seqs)):
# Update segment in idpair_to_segments
if len(aln_str) < MIN_LEN:
continue
new_bp_covered = len(aln_str) - aln_str.count("-")
# print(f"Extending {covered_seg} by {new_bp_covered}")
new_bp.append(new_bp_covered)
new_seq = aln_str
if covered_seg.strand == 1:
new_seg = Segment(covered_seg.idp, uncovered_seg.start, uncovered_seg.start + new_bp_covered, covered_seg.strand)
if new_bp_covered > 0:
segs[seg_idx] = Segment(covered_seg.idp, covered_seg.start, new_seg.stop, covered_seg.strand)
else:
aln_str = Seq(aln_str).reverse_complement()
new_seg = Segment(covered_seg.idp, covered_seg.start - new_bp_covered, covered_seg.start, covered_seg.strand)
if new_bp_covered > 0:
segs[seg_idx] = Segment(covered_seg.idp, new_seg.start, covered_seg.stop, covered_seg.strand)
new_record = SeqRecord(
seq=new_seq,
id=f"cluster{cluster_idx} s{gid_to_cid_to_index[covered_seg.idp.gid][covered_seg.idp.cid]}:p{new_seg.start if new_seg.strand == 1 else new_seg.stop}",
name=gid_to_index[covered_seg.idp.gid],
annotations={"start": new_seg.start, "end": new_seg.stop, "strand": new_seg.strand}
)
# if covered_seg.strand == 1:
new_lcb.append(new_record)
if len(new_lcb) > 0:
ret_lcbs.append(new_lcb)
return ret_lcbs
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