Flair correct crashing on one specific read conversion on hsa chr7; v3.0.0.b1

[semmrich]

We are running an ONT long-read RNA-Seq cohort of 82 samples with matching short-read RNA-Seq data to produce a FLAIR transcriptome. Below the center code of the sbatch script:

flair align \
  -r "$ONT_FASTQ" \
  -g "$GENOME" \
  -o "${OD}/${SAMPLE}" \
  -t ${SLURM_CPUS_PER_TASK:-8}

flair correct \
  -q "${OD}/${SAMPLE}.bed" \
  -f "$GTF" \
  --junction_tab "$SJ_TAB" \
  -o "${OD}/${SAMPLE}" \
  -t ${SLURM_CPUS_PER_TASK:-8}

One sample crashed on one bad line in a temp.bed file, below the complete error msg:

total alignments in bam file (includes unaligned reads): 14594009
total alignments in bam file (includes unaligned reads): 14594009
total non-secondary alignments: 14594009
total non-secondary alignments: 14594009
total primary alignments with quality >= 0: 13316995
total primary alignments with quality >= 0: 13316995
total supplementary alignments with quality >= 0: 1277014
total supplementary alignments with quality >= 0: 1277014
Flair align took 5:17:28
Flair align took 5:17:28
Read skipped: Conversion of 1cbacdf8-8a75-4f57-a94d-c5bf70a940ea chr7:44961701-44961929 to junctions failed
Error: juncsToBed12 ss1/ss2. Please report problem at https://github.com/BrooksLabUCSC/flair/issues
multiprocessing.pool.RemoteTraceback: 
"""
Traceback (most recent call last):
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/site-packages/flair/ssPrep.py", line 430, in ssPrep
    correctReads(readsBed, intervalTree, junctionBoundaryDict, chrom, resolveStrand, workingDir, chrom, errFile)
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/site-packages/flair/ssPrep.py", line 308, in correctReads
    minSize = min(sizes)
              ^^^^^^^^^^
TypeError: 'NoneType' object is not iterable

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/pool.py", line 125, in worker
    result = (True, func(*args, **kwds))
                    ^^^^^^^^^^^^^^^^^^^
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/site-packages/flair/ssPrep.py", line 432, in ssPrep
    raise Exception("** correctReads FAILED for %s" % (readsBed)) from ex
Exception: ** correctReads FAILED for /home/storage/projects/isoforms/ens115_assembly/FLAIR_v3/6405d1f8-a413-446c-8fa2-2bb9c37c1710/chr7_temp_reads.bed
"""

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/site-packages/flair/flair_cli.py", line 85, in main
    flair_module_run(opts, args.module, args.module_args)
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/site-packages/flair/flair_cli.py", line 52, in flair_module_run
    correct()
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/site-packages/flair/flair_correct.py", line 175, in correct
    for i in p.imap(ssPrep,cmds):
             ^^^^^^^^^^^^^^^^^^^
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/pool.py", line 873, in next
    raise value
Exception: ** correctReads FAILED for /home/storage/projects/isoforms/ens115_assembly/FLAIR_v3/6405d1f8-a413-446c-8fa2-2bb9c37c1710/chr7_temp_reads.bed
Exception ignored in: <function Pool.__del__ at 0x7f0bb0dab240>
Traceback (most recent call last):
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/pool.py", line 271, in __del__
    self._change_notifier.put(None)
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/queues.py", line 399, in put
    self._writer.send_bytes(obj)
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/connection.py", line 200, in send_bytes
    self._send_bytes(m[offset:offset + size])
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/connection.py", line 427, in _send_bytes
    self._send(header + buf)
  File "/home/storage/resources/tools/flair_3.0.0b1/lib/python3.12/multiprocessing/connection.py", line 384, in _send
    n = write(self._handle, buf)
        ^^^^^^^^^^^^^^^^^^^^^^^^
OSError: [Errno 9] Bad file descriptor

Interestingly, when running FLAIR v2.0.0 the same sample mate pair run through just fine.

Below I have peeked the temp.bed file in question and grepped the bad line just FYI:

head ~/debug/chr7_temp_reads.bed 
chr7	22458	26167	7d85b770-6d81-43a2-8da2-be85dfeb6576	1	-	22458	26167	217,95,2	21520,1733,	0,1976,
chr7	23597	25308	6687fce4-fed3-4a1d-8117-b196cc47eeab	1	+	23597	25308	27,158,119	2413,845,	0,866,
chr7	27533	27733	fba4a588-fc89-4296-abd4-c846680e78e5	0	-	27533	27733	99,99,99	1200,	0,
chr7	27533	27733	60490037-541c-40fb-95ae-05ce47027a44	0	-	27533	27733	99,99,99	1200,	0,
chr7	28427	30322	75ad9edc-0a54-4acc-9aea-a67b42b88a9a	0	+	28427	30322	99,99,99	11895,	0,
chr7	31059	31248	31239e31-0b72-4d38-9c5e-c11d80320754	0	-	31059	31248	99,99,99	1189,	0,
chr7	33565	33753	f709bf34-d8fc-48ce-9b61-823bc3acbf63	28	-	33565	33753	99,99,99	1188,	0,
chr7	35091	35286	8c2ab4eb-5f0a-40c7-8738-0f84b7699b4d	11	+	35091	35286	99,99,99	1195,	0,
chr7	35091	36580	8abb608c-654d-489b-b776-ffff90648765	1	+	35091	36580	27,158,119	3195,205,445,	0,724,1044,
chr7	35377	36569	a2ddeddd-f164-4946-80cd-0f5fe4b32e55	3	-	35377	36569	99,99,99	11192,	0,

grep '1cbacdf8-8a75-4f57-a94d-c5bf70a940ea' /home/storage/projects/isoforms/ens115_assembly/FLAIR_v3/00105P01/isoforms/00105P01.bed
chr7	44961701	44961929	1cbacdf8-8a75-4f57-a94d-c5bf70a940ea	60	+	44961701	44961929	99,99,99	2	19,179,	0,49,

tail ~/debug/chr7_temp_reads.bed 
chr7	158976705	158977372	ca9f40a5-9b94-4784-a64a-983168913987	60	+	158976705	158977372	99,99,99	1	667,	0,
chr7	158976705	158977372	d39f28d3-30bc-4c73-aabd-e7087e955eb4	60	+	158976705	158977372	99,99,99	1	667,	0,
chr7	158976705	158977375	2c96b8d4-0d1d-4279-9449-df99b80aa563	60	+	158976705	158977375	99,99,99	1	670,	0,
chr7	158976709	158977380	2da1bf53-7d14-4a29-91d9-f462b0660f70	60	+	158976709	158977380	99,99,99	1	671,	0,
chr7	158976725	158977374	f12082cd-9448-4d83-ba09-828ff9bd441a	60	-	158976725	158977374	99,99,99	1	649,	0,
chr7	158976935	158977022	f6080973-4ded-4372-b34b-2ed3669ef80a	21	+	158976935	158977022	99,99,99	1	87,	0,
chr7	158976942	158977326	bcfcd96d-3ca5-4df2-a263-ed93d128bb78	60	-	158976942	158977326	99,99,99	1	384,	0,
chr7	158977108	158977450	f28f606a-1c2c-4045-b649-ffe02ee865e4	60	+	158977108	158977450	99,99,99	1	342,	0,
chr7	158977119	158977885	8b24eae8-b8cf-4e1a-a67b-5c330478ba36	60	+	158977119	158977885	27,158,119	2	24,608,	0,158,
chr7	159070336	159070598	2ce8e690-75ca-496a-827c-9917e2918543	60	+	159070336	159070598	99,99,99	1	262,	0,

Hope this helps.

FLAIR is awesome, Thx so much for this superb tool!

Viele Grüße aus ffm Deutschland :)