Filter fusion artifacts (resolves #29)

resolves #29
related to BD2KGenomics/protect#237

Fusion calling often has FPS related to homology, especially among
mitochondrial  and (lnc,etc) RNA genes. Furthermore, transcriptional
readthroughs are usually oncologically irrelevant. This PR adds
filtering flags for mitochondrial, Immunoglobulin, RP11- (lncRNA) and
transcriptional readthrough filtering.

fusion.py

@@ -20,6 +20,15 @@
 genetic_code = {''.join([b1, b2, b3]): aa
  for aa, b1, b2, b3 in zip(amino, base1, base2, base3)}
 
+BEDPE = collections.namedtuple('BEDPE',
+ 'chrom1, start1, end1, '
+ 'chrom2, start2, end2, '
+ 'name, '
+ 'score, '
+ 'strand1, strand2, '
+ 'junctionSeq1, junctionSeq2, '
+ 'hugo1, hugo2')
+
 
 def get_transcriptome_data(infile):
  """
@@ -50,17 +59,20 @@ def get_transcriptome_data(infile):
  return transcript_cds, gene_transcripts
 
 
-def get_exons(genome_file, annotation_file):
+def get_exons(genome_file, annotation_file, fusions):
  """
  Generates list of GTFRecord objects for each transcript
 
  :param file genome_file: Reference genome FASTA file
  :param file annotation_file: Genome annotation file (GTF)
+ :param list(BEDPE) fusions: The fusions under consideration for this sample.
  :return: GTFRecord exons
  :rtype: dict
  """
+ annotation_file.seek(0)
  chroms ={}
  exons = collections.defaultdict(list)
+ genes_of_interest = set(sum([(record.hugo1, record.hugo2) for record in fusions], ()))
  for header, comment, seq in read_fasta(genome_file, 'ACGTN'):
  chroms[header] = seq
 
@@ -69,12 +81,31 @@ def get_exons(genome_file, annotation_file):
  continue
  else:
  gtf = GTFRecord(line)
- if gtf.feature == 'exon':
+ if gtf.feature == 'exon' and gtf.gene_name in genes_of_interest:
  gtf.sequence = chroms[gtf.seqname][gtf.start - 1: gtf.end]
  exons[gtf.transcript_id].append(gtf)
  return exons
 
 
+def read_genes_from_gtf(gtf_file):
+ """
+ Read the gene annotations into a dict
+
+ :param file gtf_file: A file handle ot the annotation file.
+ :returns: A dict of a gtf record for each gene
+ :rtype: dict(string, GTFRecord)
+ """
+ gene_annotations = {}
+ for line in gtf_file:
+ if line.startswith('#'):
+ continue
+ else:
+ gtf = GTFRecord(line)
+ if gtf.feature == 'gene':
+ gene_annotations[gtf.gene_name] = gtf
+ return gene_annotations
+
+
 def translate(seq):
  """
  Translates DNA sequence into protein sequence using globally defined genetic code
@@ -93,13 +124,52 @@ def translate(seq):
  return ''.join(protein)
 
 
-def read_fusions(fusion_file):
+def rna_gene_in_bedpe(record):
+ """
+ Determine if one of the two candidates in a BEDPE line is an rna gene.
+
+ :param BEDPE record: A BEDPE line from the input file
+ :returns: True if one of the candidates is an RNA gene and False if not
+ :rtype: bool
+ """
+ # These are heuristics
+ return 'RP11-' in record.hugo1 or 'RP11-' in record.hugo2
+
+
+def readthrough_in_bedpe(record, annotation, rt_threshold):
+ """
+ Determine if the two genes in the record are within `rt_threshold` bp of each other on the same
+ chromosome.
+
+ :param BEDPE record: A BEDPE line from the input file
+ :param dict(str, GTFRecord) annotation: see `read_fusions:gene_annotations`
+ :param rt_threshold: The genomic distance on the same chromosome below which we will call a
+ candidate fusion a readthrough.
+ :returns: True if the pair is considered a readthrough and False if not
+ :rtype: bool
+ """
+ return (record.chrom1 == record.chrom2 and
+ ((annotation[record.hugo1].start <= annotation[record.hugo2].start <=
+ annotation[record.hugo1].end + rt_threshold) or
+ (annotation[record.hugo2].start <= annotation[record.hugo1].start <=
+ annotation[record.hugo2].end + rt_threshold)))
+
+
+def read_fusions(fusion_file, gene_annotations, filter_mt, filter_ig, filter_rg, filter_rt,
+ rt_threshold):
  """
  Reads in gene fusion predictions in modified BEDPE format.
  In addition to the basic BEDPE features, this function requires the fusion
  junction sequences and HUGO names for the donor and acceptor genes.
 
+ :param filter_ig:
  :param file fusion_file: Fusion calls in BEDPE format
+ :param dict(str, GTFRecord) gene_annotations: The gene annotations form the gtf
+ :param bool filter_mt: Filter mitochondrial events?
+ :param bool filter_ig: Filter immunoglobulin pairs?
+ :param bool filter_rg: Filter RNA-Gene events?
+ :param bool filter_rt: Filter transcriptional read-throughs?
+ :param int rt_threshold: Distance threshold to call a readthrough
  :returns: list of BEDPE namedtuples
  :rtype: list
 
@@ -119,31 +189,45 @@ def read_fusions(fusion_file):
  hugo1: HUGO name for first feature
  hugo2: HUGO name for second feature
  """
- BEDPE = collections.namedtuple('BEDPE',
- 'chrom1, start1, end1, '
- 'chrom2, start2, end2, '
- 'name, score, '
- 'strand1, strand2, '
- 'junctionSeq1, junctionSeq2, '
- 'hugo1, hugo2')
 
  calls = []
+
  for line in csv.reader(fusion_file, delimiter='\t'):
  if line[0].startswith('#'):
  continue
  try:
- calls.append(BEDPE(*line))
-
+ record = BEDPE(*line)
  except TypeError:
  raise ValueError("ERROR: fusion file is malformed.\n{}".format(read_fusions.__doc__))
 
+ if filter_mt and 'M' in record.chrom1 or 'M' in record.chrom2:
+ logging.warning("Rejecting %s-%s for containing a Mitochondrial gene.", record.hugo1,
+ record.hugo2)
+ continue
+ elif filter_ig and record.hugo1.startswith('IG') and record.hugo2.startswith('IG'):
+ logging.warning("Rejecting %s-%s an an Immunoglobulin gene pair.", record.hugo1,
+ record.hugo2)
+ continue
+ elif filter_rg and rna_gene_in_bedpe(record):
+ logging.warning("Rejecting %s-%s for containing an RNA gene.", record.hugo1,
+ record.hugo2)
+ continue
+ elif filter_rt and readthrough_in_bedpe(record, gene_annotations, rt_threshold):
+ logging.warning("Rejecting %s-%s as a potential readthrough.", record.hugo1,
+ record.hugo2)
+ continue
+ else:
+ logging.info("Accepting %s-%s for further study.", record.hugo1, record.hugo2)
+ calls.append(record)
+
  return calls
 
 # Namedtuple for storing alignment metrics
-# Neeeds to be global for pickling
+# Needs to be global for pickling
 AlignStats = collections.namedtuple('AlignStats',
  'qstart, qstop, rstart, rstop, insertions, deletions')
 
+
 def align_filter(ref, query, mode, mismatches_per_kb=1):
  """
  Aligns query to reference CDS sequence using the Smith-Waterman algorithm.

test/test_transgene.py

@@ -107,6 +107,10 @@ def _test_transgene(self, use_RNA=False, use_DNA=False, fusions=False):
  'annotation.gtf')) if fusions else None
  params.genome_file = open(self._get_input_path('test_input/chr21.fa')) if fusions else None
 
+ params.filter_mt = params.filter_rg = params.filter_rt = False
+ params.rt_threshold = 100000
+ params.rg_file = None
+
  params.cores = 3
  transgene.main(params)
  output = {'9mer': {'fasta': 'unit_test_tumor_9_mer_snpeffed.faa',

transgene.py

@@ -26,7 +26,11 @@
 import sys
 
 from common import read_fasta, chrom_sort
-from fusion import get_transcriptome_data, read_fusions, insert_fusions, get_exons
+from fusion import (get_exons,
+ get_transcriptome_data,
+ insert_fusions,
+ read_fusions,
+ read_genes_from_gtf)
 
 
 # A named tuple describing the result of running reject_mutation on a mutation.
@@ -1036,15 +1040,13 @@ def main(params):
  transcriptome, gene_transcript_ids = None, None
 
  # Load data from fusion file
+ fusions = exons = None
  if params.fusion_file:
- fusions = read_fusions(params.fusion_file)
- else:
- fusions = None
-
- if params.genome_file and params.annotation_file:
- exons = get_exons(params.genome_file, params.annotation_file)
- else:
- exons = None
+ gene_annotations = read_genes_from_gtf(params.annotation_file)
+ fusions = read_fusions(params.fusion_file, gene_annotations, params.filter_mt,
+ params.filter_ig, params.filter_rg, params.filter_rt,
+ params.rt_threshold)
+ exons = get_exons(params.genome_file, params.annotation_file, fusions)
 
  for peplen in params.pep_lens.split(','):
  logging.info('Processing %s-mers', peplen)
@@ -1057,7 +1059,8 @@ def main(params):
  if proteome_data and snvs:
  insert_snvs(proteome_data, snvs, tumfile, normfile, int(peplen), params.rna_file)
  if transcriptome and gene_transcript_ids and fusions:
- insert_fusions(transcriptome, fusions, gene_transcript_ids, int(peplen), tumfile, exons=exons)
+ insert_fusions(transcriptome, fusions, gene_transcript_ids, int(peplen), tumfile,
+ exons=exons)
 
  if params.no_json_dumps:
  shutil.move(tumfile_path, '_'.join([params.prefix, 'tumor', peplen,
@@ -1080,36 +1083,36 @@ def run_transgene():
  This will try to run transgene from system arguments
  """
  parser = argparse.ArgumentParser(description=main.__doc__)
- parser.add_argument('--peptides', dest='peptide_file',
-   type=argparse.FileType('r'),
+ # SNV related options
+ parser.add_argument('--peptides', dest='peptide_file', type=argparse.FileType('r'),
  help='Path to GENCODE translation FASTA file')
- parser.add_argument('--transcripts', dest='transcript_file',
- type=argparse.FileType('r'),
- help='Path to GENCODE transcript FASTA file')
- parser.add_argument('--snpeff', dest='snpeff_file',
- type=argparse.FileType('r'),
+ parser.add_argument('--snpeff', dest='snpeff_file', type=argparse.FileType('r'),
  help='Path to snpeff file')
- parser.add_argument('--cores', dest='cores', type=int,
- help='Number of cores to use for the filtering step.', required=False,
- default=1)
- parser.add_argument('--fusions', dest='fusion_file',
- help='Path to gene fusion file',
+
+ # Fusion related options
+ parser.add_argument('--fusions', dest='fusion_file', help='Path to gene fusion file',
  type=argparse.FileType('r'))
+ parser.add_argument('--transcripts', dest='transcript_file', type=argparse.FileType('r'),
+ help='Path to GENCODE transcript FASTA file. Required if calling fusions.')
  parser.add_argument('--genome', dest='genome_file',
- help='Path to reference genome file',
+ help='Path to reference genome file, Required if calling fusions.',
  type=argparse.FileType('r'))
  parser.add_argument('--annotation', dest='annotation_file',
- help='Path to gencode annotation file',
+ help='Path to gencode annotation file. Required if calling fusions.',
  type=argparse.FileType('r'))
- parser.add_argument('--prefix', dest='prefix', type=str,
- help='Prefix for output file names', required=True)
- parser.add_argument('--pep_lens', dest='pep_lens', type=str,
- help='Desired peptide lengths to process. '
- 'The argument should be in the form of comma separated values. '
- 'E.g. 9,15', required=False, default='9,10,15')
- parser.add_argument('--no_json_dumps', action='store_true',
- help='Do not educe peptide fasta record names in the output by dumping the '
- 'mapping info into a .map json file.', required=False, default=False)
+ parser.add_argument('--filter_mt_fusions', dest='filter_mt', action='store_true',
+ help='Filter fusions involving Mitochondrial genes.', required=False)
+ parser.add_argument('--filter_ig_pairs', dest='filter_ig', action='store_true',
+ help='Filter fusions involving two immunoglobulin genes (IGXXX).',
+ required=False)
+ parser.add_argument('--filter_rna_gene_fusions', dest='filter_rg', action='store_true',
+ help='Filter fusions involving RNA genes (RP11-XXXX).', required=False)
+ parser.add_argument('--filter_readthroughs', dest='filter_rt', action='store_true',
+ help='Filter transcriptional read-troughs.', required=False)
+ parser.add_argument('--readthrough_threshold', dest='rt_threshold', type=int,
+ help='Genomic distance between candidates on the same strand below which a '
+ 'fusion will be considered a read-through.', default=500000, required=False)
+
  # RNA-Aware options
  parser.add_argument('--rna_file', dest='rna_file', help='The path to an RNA-seq bam file. If '
  'provided, the vcf will be filtered for coding mutations only. The file '
@@ -1122,6 +1125,7 @@ def run_transgene():
  parser.add_argument('--min_rna_alt_freq', dest='rna_min_alt_freq', help='The ALT allele '
  'frequency (as a fraction) in the RNA-Seq below which we will reject the '
  'mutation.', type=float, required=False, default=0.1)
+
  # OxoG filtering options
  parser.add_argument('--filterOxoG', dest='filter_oxog', action='store_true', help='Filter the '
  'calls for OxoG artifacts. This feature requires a tumor dna bam as input.',
@@ -1133,19 +1137,38 @@ def run_transgene():
  'allele frequency (as a fraction) in the DNA-Seq below which we will flag'
  'the mutation as being an OxoG variant.',
  type=float, required=False, default=0.1)
+
+ # Logging
  parser.add_argument('--log_level', dest='log_level', help='The level of logging above which '
  'messages should be printed.', required=False, choices={'DEBUG', 'INFO',
  'WARNING', 'ERROR'},
  default='INFO')
  parser.add_argument('--log_file', dest='logfile', help='A path to a logfile.', type=str,
  required=False, default=None)
+
+ # Misc
+ parser.add_argument('--prefix', dest='prefix', type=str, help='Prefix for output file names.',
+ required=True)
+ parser.add_argument('--pep_lens', dest='pep_lens', type=str, help='Desired peptide lengths to '
+ 'process. The argument should be in the form of comma separated values. '
+ 'E.g. 9,15', required=False, default='9,10,15')
+ parser.add_argument('--no_json_dumps', action='store_true',
+ help='Do not educe peptide fasta record names in the output by dumping the '
+ 'mapping info into a .map json file.', required=False, default=False)
+ parser.add_argument('--cores', dest='cores', type=int,
+ help='Number of cores to use for the filtering step.', required=False,
+ default=1)
  params = parser.parse_args()
 
  if params.snpeff_file and not params.peptide_file:
  raise ValueError('VCF file requires GENCODE translation FASTA file')
 
  if params.fusion_file and not params.transcript_file:
- raise ValueError('Fusion file requires GENCODE transcripts FASTA file')
+ raise ValueError('Fusion calling requires GENCODE transcripts FASTA file')
+ if params.fusion_file and not params.annotation_file:
+ raise ValueError('Fusion calling requires GENCODE gtf annotation file')
+ if params.fusion_file and not params.genome_file:
+ raise ValueError('Fusion calling requires genomic fasta')
 
  if params.filter_oxog:
  if not params.dna_file: