def read_gff(self, line, prefix):
# Takes prefix b/c reader returns comments, invalids, ignored
# and this method writes them to output files
# That's kind of messy
gffreader = GFFReader()
reader = open(line, 'rb')
genes, comments, invalids, ignored = gffreader.read_file(reader)
for gene in genes:
self.add_gene(gene)
# Write comments, invalid lines and ignored features
with open(prefix + "/genome.comments.gff", 'w') as comments_file:
for comment in comments:
comments_file.write(comment)
with open(prefix + "/genome.invalid.gff", 'w') as invalid_file:
for invalid in invalids:
invalid_file.write(invalid)
with open(prefix + "/genome.ignored.gff", 'w') as ignored_file:
for item in ignored:
ignored_file.write(item)
def read_gff(self, line, prefix):
# Takes prefix b/c reader returns comments, invalids, ignored
# and this method writes them to output files
# That's kind of messy
gffreader = GFFReader()
reader = open(line, 'rb')
genes, comments, invalids, ignored = gffreader.read_file(reader)
for gene in genes:
self.add_gene(gene)
# Write comments, invalid lines and ignored features
with open(prefix + "/genome.comments.gff", 'w') as comments_file:
for comment in comments:
comments_file.write(comment)
with open(prefix + "/genome.invalid.gff", 'w') as invalid_file:
for invalid in invalids:
invalid_file.write(invalid)
with open(prefix + "/genome.ignored.gff", 'w') as ignored_file:
for item in ignored:
ignored_file.write(item)
def read_gff(self, line):
gffreader = GFFReader()
reader = open(line, 'rb')
genes = gffreader.read_file(reader)
for gene in genes:
self.add_gene(gene)
def main():
print("Yarr! I be Cap'n Primer.\n")
# Check dependencies
print("Ahoy, let's check these scurvy dependencies...")
check_dependencies()
# Verify files
print("Yarr! Now to verify the input files...")
target_fasta_path = 'targets.fasta'
genome_fasta_path = 'genome.fasta'
gff_path = 'genome.gff'
excluded_primers_path = 'primers_to_exclude.boulder-io'
options_path = 'primer3_options'
for path in [target_fasta_path, genome_fasta_path, excluded_primers_path, options_path]:
found_it = verify_path(path)
if not found_it:
exit()
print("Shiver me timbers, the input files be present. Now I'll be reading them. Savvy?")
# Read files
print("Reading the scurvy " + target_fasta_path + " file...")
fasta_reader = FastaReader()
with open(target_fasta_path, 'rb') as target_file:
target_seqs = fasta_reader.read(target_file)
if not target_seqs:
print("Yarr! Error reading target fasta. Walk the plank. " + random_insult() + "\n")
sys.exit()
cds_segment_lengths = None
if verify_path(gff_path):
gff_reader = GFFReader()
print("Reading the scurvy " + gff_path + " file...")
with open(gff_path, 'rb') as gff_file:
cds_segment_lengths = gff_reader.read(gff_file)
if not cds_segment_lengths:
print("Yarr! Error reading GFF! Walk the plank. " + random_insult() + "\n")
sys.exit()
boulder_io_reader = BoulderIOReader()
print("Reading the scurvy " + excluded_primers_path + " file...")
with open(excluded_primers_path, 'rb') as exclude_file:
primers_to_exclude = boulder_io_reader.read_primer3_output(exclude_file)
if not primers_to_exclude:
print("Yarr! Error reading excluded primers file. Walk the plank. " + random_insult() + "\n")
primer3_options = ""
print("Reading the scurvy " + options_path + " file...")
with open(options_path, 'rb') as options_file:
for line in options_file:
primer3_options += line
print("")
# Prepare input for primer3_core
print("Yarr! Preparing input for primer3_core...")
boulder_formatter = BoulderIOFormatter()
if cds_segment_lengths:
boulder_formatter.segment_lengths = cds_segment_lengths
print("Yarr! Writing input file for primer3_core...")
with open("target_seqs.boulder-io", "wb") as boulderfile:
# write config data first
boulderfile.write(primer3_options)
exclude_entries = []
for seq in target_seqs:
for primer in primers_to_exclude:
if primer.target_sequence.header == seq.header:
exclude_entries.append(primer.to_excluded_region_entry())
boulderfile.write(boulder_formatter.format_seq(seq, exclude_entries))
exclude_entries[:] = []
# Run primer3_core
print("Yarr! Running primer3_core!")
os.system("primer3_core < target_seqs.boulder-io > primers.boulder-io")
print("Yarr! Ran primer3_core!")
# Verify
if file_is_empty("primers.boulder-io"):
print("Yarr! No output from primer3_core! Walk the plank. " + random_insult() + "\n")
sys.exit()
# Read primers from file
print("Yarr! Reading output from primer3_core!")
with open("primers.boulder-io", "rb") as primersfile:
primers = boulder_io_reader.read_primer3_output(primersfile)
# Verify again, just for fun
if not primers:
print("Yarr! No primers in the scurvy primers.boulder-io file! Walk the plank. " + random_insult() + "\n")
sys.exit()
print("Yarr! We got " + str(len(primers)) + " scurvy primers!")
# Convert primers.boulder-io file to left and right primer seqs, then write to fasta
print("Yarr! Making scurvy fasta files of the left and right primers from the" +
" primer3_core output. Shiver me timbers!")
with open("left_right_primers.fasta", "wb") as primersfasta:
for primer in primers:
primersfasta.write(primer.left_primer_to_fasta())
primersfasta.write(primer.right_primer_to_fasta())
# Verify that file was written
if file_is_empty("left_right_primers.fasta"):
print("Yarr! Left and right primers failed to write! Walk the plank. " + random_insult() + "\n")
sys.exit()
# BLAST PRIMER SEQUENCES AGAINST GENOME TO MAKE SURE THEY ONLY AMPLIFY ONE REGION
print("Yarr! Preparing blast database!")
# makeblastdb -in Bdor.Trinity.reallyfiltered.fasta -dbtype nucl
os.system("makeblastdb -in genome.fasta -dbtype nucl > /dev/null")
# Verify that the database was created
if file_is_empty("genome.fasta.nhr"):
print("Yarr! Database wasn't created. Walk the plank. " + random_insult() + "\n")
sys.exit()
print("Yarr! Running blast on those scurvy left and right primers...")
os.system('blastall -p blastn -d genome.fasta -i left_right_primers.fasta '+
'-r 1 -q 1 -G 1 -E 2 -W 9 -F "m D" -U -m 9 -b 4 > left_right_primers.blastout')
# Verify that blast produced results
if file_is_empty("left_right_primers.blastout"):
print("Yarr! No output from blast! Walk the plank. " + random_insult() + "\n")
sys.exit()
print("Yarr! Blast finished running.")
# Read in blast output
blast_parser = BlastOutputParser()
with open("left_right_primers.blastout", "rb") as blastout:
blast_results = blast_parser.parse(blastout)
if not blast_results or blast_results.number_of_hits == 0:
print("Yarr! Scurvy error reading blast results. Walk the plank. " + random_insult() + "\n")
sys.exit()
print("Yarr! We got " + str(blast_results.number_of_hits()) + " hits!")
print("Yarr! Time to filter these scurvy blast hits.")
# filter hits for each using e-value and alignment length cutoffs
MAX_E_VALUE = 1.0
MIN_ALIGNMENT_LENGTH = 12
blast_results.filter_results(MAX_E_VALUE, MIN_ALIGNMENT_LENGTH)
print("Yarr! Now we got " + str(blast_results.number_of_hits()) + " hits!")
# Verify that for a given primer, the left and right each map to exactly one common region in the genome
one_match_primers = []
for primer in primers:
left_primer_name = primer.target_sequence.header
left_primer_name += "_" + primer.primer_name + "_left"
right_primer_name = primer.target_sequence.header
right_primer_name += "_" + primer.primer_name + "_right"
if blast_results.number_of_common_hits(left_primer_name, right_primer_name) == 1:
one_match_primers.append(primer)
print("Yarr! There be " + str(len(one_match_primers)) + " one-match primers. Yarr!")
# TODO check that it's the right region????
# Write those primers to fasta
with open("verified_primers.fasta", "wb") as primfasta:
for primer in one_match_primers:
primfasta.write(primer.to_fasta())
# Verify that fasta
if file_is_empty("verified_primers.fasta"):
print("Yarr! Failed to write verified primers to fasta! Walk the plank. " + random_insult() + "\n")
sys.exit()
print("Yarr! Wrote verified primers to fasta.")
# Write those primers to a tsv
write_primer_table("verified_primers.tsv", one_match_primers)
# Verify that tsv
if file_is_empty("verified_primers.tsv"):
print("Yarr! Failed to write verified primers to tsv! Walk the plank. " + random_insult() + "\n")
sys.exit()
print("Yarr! Wrote verified primers to tsv.")