def test_too_far_away(self):
""" A case where the NCSJ should not be corrected because it is too far
away from the closest annotated junction relative to the maxDist
parameter.
Toy transcript with sequence A|GAA, where the splice motif
is noncanonical.
chr1: 23,071,357 - 23,072,126
"""
# Process references
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test_jns/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
genome = Fasta("input_files/hg38_chr1.fa")
# Init transcript object
sam_fields = [
"test_read", "0", "chr1", "23071357", "255", "1M766N3M", "*", "0",
"0", "AGAA", "*", "NM:i:0", "MD:Z:6"
]
transcript = t2.Transcript(sam_fields, genome, sjAnnot)
jnNumber = 0
maxDist = 1
correction_status, reason, dist = TC.attempt_jn_correction(
transcript, jnNumber, genome, donors, acceptors, sjAnnot, maxDist)
assert correction_status == False
assert reason == "TooFarFromAnnotJn"
assert dist == 2
def test_crash(self):
""" This is a Drosophila junction that borders a small match preceded by
a 7 bp deletion. It is supposed to crash correction, which will result
in a categorization of 'Other' in the log """
# Process references
sjFile = "input_files/drosophila_example/chr3R_SJs.tsv"
outprefix = "scratch/dmel_crash/"
tmp_dir = "scratch/dmel_crash/TC_tmp/"
chroms = set(["chr3R"])
donors, acceptors, sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
genome = Fasta("input_files/drosophila_example/chr3R.fa")
# Init transcript object
sam_fields = [
"test_read", "0", "chr3R", "14890420", "255", "7M7D2M264N7M", "*",
"0", "0", "GATCAAACAACAAGTC", "*"
]
transcript = t2.Transcript(sam_fields, genome, sjAnnot)
jnNumber = 0
maxDist = 5
# Attempt to correct the splice junction
correction_status, reason, dist = TC.attempt_jn_correction(
transcript, jnNumber, genome, donors, acceptors, sjAnnot, maxDist)
assert correction_status == False
assert reason == "Other"
assert dist == 5
def test_noncanonical(self):
""" Transcript should be noncanonical and un-annotated prior to
correction, but be canonical and annotated afterwards """
sam = "input_files/sams/deletion_insertion_mismatch_nc.sam"
sjFile = "input_files/GM12878_SJs_chr1.tab"
tmp_dir = "scratch/test_jIjM/TC_tmp/"
chroms = set(["chr1"])
refs = dstruct.Struct()
refs.genome = Fasta("input_files/hg38_chr1.fa")
refs.donors, refs.acceptors, refs.sjAnnot = TC.processSpliceAnnotation(sjFile, tmp_dir,
chroms)
with open(sam, 'r') as f:
sam_line = f.readline().strip()
transcript, logInfo = TC.transcript_init(sam_line, refs.genome,
refs.sjAnnot)
assert transcript.allJnsAnnotated == False
assert transcript.isCanonical == False
# Now correct the junction and retest
upd_transcript, TE = TC.cleanNoncanonical(transcript, refs, 5, logInfo)
assert upd_transcript.allJnsAnnotated == True
assert upd_transcript.isCanonical == True
def test_fix_donor_case3(self):
""" Toy transcript with sequence AAGGT|GAA, where the splice motif
is noncanonical but located 2 bp from a canonical splice donor.
chr1: 23,071,357 - 23,072,126
So-called case #3
"""
# Process references
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjDict = TC.processSpliceAnnotation(sjFile, tmp_dir,
chroms)
genome = Fasta("input_files/hg38_chr1.fa")
# Init transcript object
sam_fields = ["test_read", "0", "chr1", "23071357", "255", "5M762N3M", "*",
"0", "0", "AAGGTGAA", "*", "NM:i:0", "MD:Z:8"]
transcript = t2.Transcript(sam_fields, genome, sjDict)
jnNumber = 0
maxDist = 5
donor = (transcript.spliceJunctions[jnNumber]).bounds[0]
# Attempt to correct the splice donor side of the junction (left)
new_seq, new_cigar = TC.fix_one_side_of_junction(transcript.CHROM,
transcript.POS, jnNumber,
donor, -2, genome,
transcript.SEQ,
transcript.CIGAR)
assert new_seq == "AAGGAA"
assert new_cigar == "3M764N3M"
def test_no_correction(self):
""" Make sure that the attributes stay the same if no correction
was performed
"""
# Process references
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donor, acceptor, sjDict = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
genome = Fasta("input_files/hg38_chr1.fa")
# Init transcript object
sam_fields = [
"test_read", "0", "chr1", "23071357", "255", "1M766N3M", "*", "0",
"0", "AGAA", "*", "NM:i:0", "MD:Z:4"
]
transcript = t2.Transcript(sam_fields, genome, sjDict)
jnNumber = 0
maxDist = 5
donor = (transcript.spliceJunctions[jnNumber]).bounds[0]
# Now test the update function
TC.update_post_ncsj_correction(transcript, jnNumber, genome, sjDict)
junction = transcript.spliceJunctions[jnNumber]
assert junction.motif_code == "0"
assert junction.isCanonical == False
assert transcript.MD == "MD:Z:4"
assert transcript.isCanonical == False
def test_find_closest_splice_acceptor_minus(self):
""" Find the closest splice acceptor, which is 1 bp downstream.
Minus strand. Note that dist is relative to the genome, not to
the direction of the transcript."""
# Process reference junctions
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjDict = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
# Intron bound info
transcriptID = "test_read"
jnNumber = 0
chrom = "chr1"
start = 22071331
end = 22073331
strand = "-"
genome = Fasta("input_files/hg38_chr1.fa")
junction = sj.SpliceJunction(transcriptID, jnNumber, chrom, start, end,
strand, genome, sjDict)
acceptor = junction.get_splice_acceptor()
closest_acceptor = TC.find_closest_bound(acceptor, acceptors)
assert closest_acceptor.start == 22071329
assert closest_acceptor.end == 22071330
assert closest_acceptor.dist == -1
def test_find_closest_sj_plus(self):
# Process reference junctions
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjDict = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
# Intron bound info
transcriptID = "test_read"
jnNumber = 0
chrom = "chr1"
start = 23071350
end = 23072124
strand = "+"
genome = Fasta("input_files/hg38_chr1.fa")
junction = sj.SpliceJunction(transcriptID, jnNumber, chrom, start, end,
strand, genome, sjDict)
closest_donor, closest_acceptor = TC.find_closest_ref_junction(
junction, donors, acceptors)
assert closest_donor.end == 23071360
assert closest_acceptor.end == 23072123
def test_find_closest_splice_donor_minus(self):
""" For a toy case with multiple donors and acceptors in close
proximity, test whether TC can find the closest reference donor
to the supplied intron bound.
Similar to before, there is an exact match for the donor, located
at 23071360 in 1-based coordinates and 23071359 in 0-based."""
# Process reference junctions
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjDict = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
# Intron bound info
transcriptID = "test_read"
jnNumber = 0
chrom = "chr1"
start = 23070360
end = 23071360
strand = "-"
genome = Fasta("input_files/hg38_chr1.fa")
junction = sj.SpliceJunction(transcriptID, jnNumber, chrom, start, end,
strand, genome, sjDict)
donor = junction.get_splice_donor()
closest_donor = TC.find_closest_bound(donor, donors)
assert closest_donor.start == 23071359
assert closest_donor.end == 23071360
assert closest_donor.dist == 0
def test_tmp_files(self):
""" Check that the expected tmp files are created."""
sj_file = "input_files/toy_sjs_mixed_chroms.txt"
chroms = set(["chr1", "chr2"])
tmp_dir = "scratch/sj_reading_test/"
os.system("mkdir -p " + tmp_dir)
donor_bt, accept_bt, annot = TC.processSpliceAnnotation(sj_file,
tmp_dir,
chroms,
process="test")
# Check if paths of tmp files are correct
assert os.path.exists(
"scratch/sj_reading_test/splice_files/test_ref_splice_donors_tmp.bed"
)
assert os.path.exists(
"scratch/sj_reading_test/splice_files/test_ref_splice_acceptors_tmp.bed"
)
assert os.path.exists(
"scratch/sj_reading_test/splice_files/test_ref_splice_donors_tmp.sorted.bed"
)
assert os.path.exists(
"scratch/sj_reading_test/splice_files/test_ref_splice_acceptors_tmp.sorted.bed"
)
def test_correct_jn(self):
""" Toy transcript with sequence A|GAA, where the splice motif
is noncanonical but located 2 bp from a canonical splice donor.
chr1: 23,071,357 - 23,072,126
"""
# Process references
sjFile = "input_files/test_junctions.txt"
outprefix = "scratch/test_jns/"
tmp_dir = "scratch/test_jns/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
genome = Fasta("input_files/hg38_chr1.fa")
# Init transcript object
sam_fields = [
"test_read", "0", "chr1", "23071357", "255", "1M766N3M", "*", "0",
"0", "AGAA", "*", "NM:i:0", "MD:Z:4"
]
transcript = t2.Transcript(sam_fields, genome, sjAnnot)
jnNumber = 0
maxDist = 5
#donor = (transcript.spliceJunctions[jnNumber]).bounds[0]
# Attempt to correct the splice junction
correction_status, reason, dist = TC.attempt_jn_correction(
transcript, jnNumber, genome, donors, acceptors, sjAnnot, maxDist)
assert correction_status == True
assert reason == "NA"
assert dist == 2
def test_find_closest_splice_acceptor_plus(self):
""" Find the closest splice acceptor, which is 17 bp upstream.
Plus strand."""
# Process reference junctions
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjDict = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
# Intron bound info
transcriptID = "test_read"
jnNumber = 0
chrom = "chr1"
start = 23071360
end = 23072140
strand = "+"
genome = Fasta("input_files/hg38_chr1.fa")
junction = sj.SpliceJunction(transcriptID, jnNumber, chrom, start, end,
strand, genome, sjDict)
acceptor = junction.get_splice_acceptor()
closest_acceptor = TC.find_closest_bound(acceptor, acceptors)
assert closest_acceptor.start == 23072122
assert closest_acceptor.end == 23072123
assert closest_acceptor.dist == -17
def test_crash_dmel(self):
""" This is a Drosophila junction that borders a small match preceded by
a 7 bp deletion. It is also supposed to crash correction, but did
not in TC v2.0.1."""
# Process references
sjFile = "input_files/drosophila_example/chr3R_SJs.tsv"
tmp_dir = "scratch/dmel/TC"
os.system("mkdir -p %s" % tmp_dir)
refs = dstruct.Struct()
chroms = set(["chr3R"])
refs.donors, refs.acceptors, refs.sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
refs.genome = Fasta("input_files/drosophila_example/chr3R.fa")
sam = "input_files/drosophila_example/no_SJ_corr.sam"
with open(sam, 'r') as f:
for sam_line in f:
if sam_line.startswith("@"):
continue
else:
sam_line = sam_line.strip().split('\t')
# Init transcript object
transcript = t2.Transcript(sam_line, refs.genome, refs.sjAnnot)
maxDist = 5
logInfo = TC.init_log_info(sam_line)
orig_CIGAR = transcript.CIGAR
orig_seq = transcript.SEQ
orig_MD = transcript.MD
expected_TE = "\t".join([
"m160713_133433_42182_c101000162550000001823232709161620_s1_p0/121139/11291_13013",
"chr3R_14890436_14890699", "NC_SJ_boundary", "5", "Uncorrected",
"Other"
]) + "\n"
assert transcript.isCanonical == False
# Attempt to correct the splice junction
new_transcript, TE_entries = TC.cleanNoncanonical(
transcript, refs, maxDist, logInfo)
print(TE_entries)
assert new_transcript.isCanonical == False
assert TE_entries == expected_TE
assert new_transcript.MD == orig_MD
assert logInfo.corrected_NC_SJs == 0
assert logInfo.uncorrected_NC_SJs == 1
assert new_transcript.CIGAR == orig_CIGAR
assert new_transcript.SEQ == orig_seq
def test_two_annotated_SJs(self):
""" Transcript with 2 junctions and each match the provided reference
"""
sam = "input_files/sams/perfectReferenceMatch_twoIntrons.sam"
genome = Fasta("input_files/hg38_chr1.fa")
sjFile = "input_files/GM12878_SJs_chr1.tab"
outprefix = "scratch/test"
tmp_dir = "scratch/test_jIjM/TC_tmp/"
chroms = set(["chr1"])
donors, acceptors, sjDict = TC.processSpliceAnnotation(sjFile, tmp_dir,
chroms)
with open(sam, 'r') as f:
sam_line = f.readline().strip().split('\t')
transcript = t2.Transcript(sam_line, genome, sjDict)
assert transcript.allJnsAnnotated == True
assert transcript.isCanonical == True
def test_splice_donors(self):
""" Make sure that the correct positions got labeled as splice donors """
sj_file = "input_files/toy_sjs_mixed_chroms.txt"
chroms = set(["chr1", "chr2"])
tmp_dir = "scratch/sj_reading_test/"
os.system("mkdir -p " + tmp_dir)
donor_bt, accept_bt, annot = TC.processSpliceAnnotation(sj_file,
tmp_dir,
chroms,
process="test")
# Remember, file is 1-based but BedTool is 0-based
expected_donors = set([99, 399])
donors = set()
for donor in donor_bt:
donors.add(donor.start)
assert donors == expected_donors
def test_crash_correction(self):
""" This is a case that is supposed to crash the NCSJ correction process,
resulting in no correction. This is because the mapping has
created a 7-bp micro-exon with a canonical but likely incorrect
junction to its left, and a non-canonical junction on its right.
Post-correction, we end up with two introns next to each other
with a zero-length exon, which is not valid."""
# Process references
sjFile = "input_files/chr11_sjs.txt"
tmp_dir = "scratch/test/TC_tmp/"
os.system("mkdir -p %s" % tmp_dir)
refs = dstruct.Struct()
chroms = set(["chr11"])
refs.donors, refs.acceptors, refs.sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
refs.genome = Fasta("input_files/hg38_chr11.fa")
sam = "input_files/sams/microexon.sam"
with open(sam, 'r') as f:
sam_line = f.readline().strip().split('\t')
# Init transcript object
transcript = t2.Transcript(sam_line, refs.genome, refs.sjAnnot)
maxDist = 5
logInfo = TC.init_log_info(sam_line)
assert transcript.isCanonical == False
# Attempt to correct the splice junction
transcript, TE_entries = TC.cleanNoncanonical(transcript, refs,
maxDist, logInfo)
orig_CIGAR = ("1211M5612N57M464N30M2717N120M1097N23M2632N146M1225N"
"140M4770N72M5051N132M1513N87M567N142M3780N100M2160N"
"59M864N31M9891N69M1711N7M1341N47M13S")
assert transcript.isCanonical == False
assert transcript.MD == "MD:Z:2473"
assert logInfo.corrected_NC_SJs == 0
assert logInfo.uncorrected_NC_SJs == 1
assert transcript.CIGAR == orig_CIGAR
def test_correct_ncsj(self):
""" Toy transcript with sequence A|GAA, where the splice motif
is noncanonical but located 2 bp from a canonical splice donor.
chr1: 23,071,357 - 23,072,126
"""
# Process references
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test_ncsj/TC_tmp/"
os.system("mkdir -p %s" % tmp_dir)
refs = dstruct.Struct()
chroms = set(["chr1"])
refs.donors, refs.acceptors, refs.sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
refs.genome = Fasta("input_files/hg38_chr1.fa")
# Init transcript object
sam_fields = [
"test_read", "0", "chr1", "23071357", "255", "1M766N3M", "*", "0",
"0", "AGAA", "*", "NM:i:0", "MD:Z:4"
]
transcript = t2.Transcript(sam_fields, refs.genome, refs.sjAnnot)
jnNumber = 0
maxDist = 5
logInfo = TC.init_log_info(sam_fields)
assert transcript.isCanonical == False
# Attempt to correct the splice junction
transcript, TE_entries = TC.cleanNoncanonical(transcript, refs,
maxDist, logInfo)
assert transcript.isCanonical == True
assert transcript.spliceJunctions[jnNumber].isCanonical == True
assert transcript.SEQ == "AAGGAA"
assert transcript.CIGAR == "3M764N3M"
assert transcript.MD == "MD:Z:6"
assert logInfo.corrected_NC_SJs == 1
def test_update(self):
""" Toy transcript with sequence A|GAA, where the splice motif
is noncanonical but located 2 bp from a canonical splice donor.
chr1: 23,071,357 - 23,072,126
"""
# Process references
sjFile = "input_files/test_junctions.txt"
tmp_dir = "scratch/test/TC_tmp/"
chroms = set(["chr1"])
donor, acceptor, sjDict = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
genome = Fasta("input_files/hg38_chr1.fa")
# Init transcript object
sam_fields = [
"test_read", "0", "chr1", "23071357", "255", "1M766N3M", "*", "0",
"0", "AGAA", "*", "NM:i:0", "MD:Z:4"
]
transcript = t2.Transcript(sam_fields, genome, sjDict)
jnNumber = 0
maxDist = 5
donor = (transcript.spliceJunctions[jnNumber]).bounds[0]
# Attempt to correct the splice donor side of the junction (left)
transcript.SEQ, transcript.CIGAR = TC.fix_one_side_of_junction(
transcript.CHROM, transcript.POS, jnNumber, donor, 2, genome,
transcript.SEQ, transcript.CIGAR)
# Now test the update function
TC.update_post_ncsj_correction(transcript, jnNumber, genome, sjDict)
junction = transcript.spliceJunctions[jnNumber]
assert junction.motif_code == "21"
assert junction.isCanonical == True
assert transcript.MD == "MD:Z:6"
assert transcript.isCanonical == True
def test_chrom_filtering(self):
""" Check that only chr1 and chr2 junctions get saved"""
sj_file = "input_files/toy_sjs_mixed_chroms.txt"
chroms = set(["chr1", "chr2"])
tmp_dir = "scratch/sj_reading_test/"
os.system("mkdir -p " + tmp_dir)
donor_bt, accept_bt, annot = TC.processSpliceAnnotation(sj_file,
tmp_dir,
chroms,
process="test")
# Check donor chroms
donor_chroms = set()
for pos in donor_bt:
donor_chroms.add(pos.chrom)
assert donor_chroms == chroms
# Check acceptor chroms
acc_chroms = set()
for pos in accept_bt:
acc_chroms.add(pos.chrom)
assert acc_chroms == chroms
def test_DIM_nc(self):
""" Correct a transcript containing a deletion, insertion, mismatch,
and noncanonical splice junction """
# Initialize options etc.
sam = "input_files/sams/deletion_insertion_mismatch_nc.sam"
genome = Fasta("input_files/hg38_chr1.fa")
sjFile = "input_files/GM12878_SJs_chr1.tab"
tmp_dir = "scratch/example/TC_tmp/"
os.system("mkdir -p %s" % tmp_dir)
chroms = set(["chr1"])
donors, acceptors, sjAnnot = TC.processSpliceAnnotation(
sjFile, tmp_dir, chroms)
outfiles = dstruct.Struct()
outfiles.TElog = open(tmp_dir + "DIM_nc_clean.TE.log", 'w')
outfiles.sam = open(tmp_dir + "DIM_nc_clean.sam", 'w')
outfiles.fasta = open(tmp_dir + "DIM_nc_clean.fasta", 'w')
outfiles.log = open(tmp_dir + "DIM_nc_clean.log", 'w')
refs = dstruct.Struct()
refs.sjAnnot = sjAnnot
refs.genome = genome
refs.donors = donors
refs.acceptors = acceptors
refs.snps = {}
refs.deletions = {}
refs.insertions = {}
options = dstruct.Struct()
options.maxLenIndel = 5
options.maxSJOffset = 5
options.correctMismatches = "true"
options.correctIndels = "true"
options.correctSJs = "true"
options.primaryOnly = True
options.canonOnly = False
# Correct the transcript
with open(sam, 'r') as f:
transcripts = [f.readline().strip()]
TC.batch_correct(transcripts, options, refs, outfiles)
# Close the output files
for handle in outfiles.values():
handle.close()
# Expected transcript attributes post-correction
correct_CIGAR = ("12M1134N126M163N202M866N74M924N191M1777N127M2109N"
"157M88N159M932N633M274N117M7696N170M1215N629M938N"
"29M428N133M254N166M390N212M253N89M163N483M")
correct_MD = "MD:Z:3709"
correct_NM = "NM:i:0"
correct_jI = (
"jI:B:i,150941429,150942562,150942689,150942851,150943054,"
"150943919,150943994,150944917,150945109,150946885,150947013,"
"150949121,150949279,150949366,150949526,150950457,150951091,"
"150951364,150951482,150959177,150959348,150960562,150961192,"
"150962129,150962159,150962586,150962720,150962973,150963140,"
"150963529,150963742,150963994,150964084,150964246")
correct_jM = "jM:B:c,21,21,21,21,21,21,21,21,21,21,21,21,21,21,21,21,21"
# Read in transcript from outfile
with open(tmp_dir + "DIM_nc_clean.sam", 'r') as f:
sam_line = f.readline().strip().split('\t')
transcript = t2.Transcript(sam_line, genome, sjAnnot)
assert transcript.CIGAR == correct_CIGAR
assert transcript.MD == correct_MD
assert transcript.NM == correct_NM
assert transcript.jI == correct_jI
assert transcript.jM == correct_jM
# Read logs and make sure they are OK
expected_log = "\t".join([
"c34150/f1p1/3707", "primary", "2", "0", "0", "1", "0", "0", "2",
"0", "1", "0"
])
with open(tmp_dir + "DIM_nc_clean.log", 'r') as f:
log = f.readline().strip()
assert log == expected_log
