flair collapse generates a confusing pysam error when nothing aligned

[diekhans]

rm -rf output/test-collapse-seg1.tmp
flair collapse -r input/seg1.cdna-ont.fastq -q expected/test-correct-seg1_all_corrected.bed -g input.tmp/genome.fa -t 4 --generate_map --temp_dir output/test-collapse-seg1.tmp --keep_intermediate -f input/annotation.gtf -o output/test-collapse-seg1 -p input/seg1.promoter-regions.bedx --annotation_reliant generate --stringent
Starting collapse...
Filtering out reads without promoter-supported TSS
Exception ignored in: <function Popen.__del__ at 0x7f37b29ad8a0>
Traceback (most recent call last):
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 624, in __del__
    self.close()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 619, in close
    self.wait()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 708, in wait
    super(Popen, self).wait()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 471, in wait
    self._wait_guts()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 464, in _wait_guts
    self._raise_if_failed()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 427, in _raise_if_failed
    raise ex
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 424, in _raise_if_failed
    raise p.procExcept
pipettor.exceptions.ProcessException: process exited 1: bedtools intersect -a output/test-collapse-seg1.tmp/tmptqxmzob2.tss.bed -b input/seg1.promoter-regions.bedx:
Error: Unable to open file input/seg1.promoter-regions.bedx. Exiting.

Making transcript fasta using annotated gtf and genome sequence
Aligning reads to reference transcripts
Counting supporting reads for annotated transcripts
Setting up unassigned reads for flair-collapse novel isoform detection
Renaming isoforms using gtf
Aligning reads to first-pass isoform reference
Aligning reads to firstpass transcripts
Counting supporting reads for firstpass transcripts
Traceback (most recent call last):
  File "/hive/users/markd/flair/projs/src/flair-markd/test/../bin/flair", line 109, in <module>
    main()
  File "/hive/users/markd/flair/projs/src/flair-markd/test/../bin/flair", line 67, in main
    [isoforms, isoform_sequences] = collapse()
                                    ^^^^^^^^^^
  File "/hive/users/markd/flair/projs/src/flair-markd/src/flair/flair_collapse.py", line 416, in collapse
    pipettor.run([mm2_cmd, count_cmd])
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/__init__.py", line 32, in run
    Pipeline(cmds, stdin=stdin, stdout=stdout, stderr=stderr, logger=logger, logLevel=logLevel).wait()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 471, in wait
    self._wait_guts()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 464, in _wait_guts
    self._raise_if_failed()
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 427, in _raise_if_failed
    raise ex
  File "/cluster/home/markd/opt/miniforge3/envs/flair-dev/lib/python3.12/site-packages/pipettor/processes.py", line 424, in _raise_if_failed
    raise p.procExcept
pipettor.exceptions.ProcessException: process exited 1: count_sam_transcripts.py --sam - -o output/test-collapse-seg1.firstpass.q.counts -t 4 --quality 1 -w 100 --stringent -i output/test-collapse-seg1.firstpass.bed --generate_map output/test-collapse-seg1.isoform.read.map.txt:
/hive/users/markd/flair/projs/src/flair-markd/test/../src/flair/count_sam_transcripts.py:127: SyntaxWarning: invalid escape sequence '\d'
  mdblocks = re.findall('\d+|\D+', md)  # ['531', '^CCAGGTGAGCCGCCCGCG', '50', 'G', '2031']
Traceback (most recent call last):
  File "/hive/users/markd/flair/projs/src/flair-markd/test/../src/flair/count_sam_transcripts.py", line 283, in <module>
    transcripttoreads = parsesam(args, transcripttoexons)
                        ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "/hive/users/markd/flair/projs/src/flair-markd/test/../src/flair/count_sam_transcripts.py", line 224, in parsesam
    samfile = pysam.AlignmentFile(args.sam, 'r')
              ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.__cinit__
  File "pysam/libcalignmentfile.pyx", line 1000, in pysam.libcalignmentfile.AlignmentFile._open
ValueError: file has no sequences defined (mode='r') - is it SAM/BAM format? Consider opening with check_sq=False

make: *** [Makefile:122: test-collapse-seg1] Error 1

[diekhans]

this also happens with an empty BED file

[diekhans]

This appears to be caused not my the invalid promoter file, but by no alignments being produced.

looks like on second pass

[diekhans]

these files are all empty

output/test-collapse-seg1.firstpass.bed                                                                       
output/test-collapse-seg1.firstpass.fa                                                                        
output/test-collapse-seg1.firstpass.unfiltered.bed