OSError: no BGZF EOF marker; file may be truncated

I have seen an issue with same names. However, I think this may be a new problem.

Using output of CellRanger 3.1.0，I’ve successfully run velocyto (version 0.17.17) with 3 samples. But I got the error information from another sample, by code

velocyto run10x /home/Script/GTE002/ /public-supool/home/Data/genes.gtf

:

Resource usage summary:

    CPU time :                                   11146.22 sec.
    Max Memory :                                 1743 MB
    Average Memory :                             1185.21 MB
    Total Requested Memory :                     -
    Delta Memory :                               -
    Max Swap :                                   -
    Max Processes :                              3
    Max Threads :                                26
    Run time :                                   11186 sec.

...
2020-03-13 13:13:45,346 - DEBUG - Read first 850 million reads
2020-03-13 13:14:01,793 - DEBUG - End of file. Reset index: start scanning from initial position.
2020-03-13 13:14:01,796 - DEBUG - 16951446 reads were skipped because no apropiate cell or umi barcode was found
2020-03-13 13:14:01,797 - DEBUG - Start molecule counting!
2020-03-13 13:14:05,705 - DEBUG - Features available for chromosomes : ['1-', '1+', '10-', '10+', '11-', '11+', '12-', '12+', '13-', '13+', '14-', '14+', '15-', '15+', '16-', '16+', '17-', '17+', '18-', '18+', '19-', '19+', '2-', '2+', '20-', '20+', '21-', '21+', '22-', '22+', '3-', '3+', '4-', '4+', '5-', '5+', '6-', '6+', '7-', '7+', '8-', '8+', '9-', '9+', 'GL000191.1-', 'GL000191.1+', 'GL000192.1-', 'GL000193.1-', 'GL000194.1-', 'GL000195.1-', 'GL000201.1-', 'GL000201.1+', 'GL000204.1-', 'GL000205.1+', 'GL000209.1+', 'GL000212.1+', 'GL000213.1-', 'GL000213.1+', 'GL000215.1-', 'GL000218.1-', 'GL000219.1-', 'GL000221.1-', 'GL000222.1-', 'GL000222.1+', 'GL000223.1-', 'GL000223.1+', 'GL000228.1-', 'GL000237.1-', 'GL000242.1+', 'MT-', 'MT+', 'X-', 'X+', 'Y-', 'Y+']
2020-03-13 13:14:05,706 - DEBUG - Mask available for chromosomes : []
2020-03-13 13:14:05,706 - DEBUG - Summarizing the results of intron validation.
2020-03-13 13:14:06,408 - DEBUG - Validated 634190 introns (of which unique intervals 204042) out of 964599 total possible introns (considering each possible transcript models).
2020-03-13 13:14:06,409 - DEBUG - Reading /home/script/GTE002/outs/cellsorted_possorted_genome_bam.bam


Traceback (most recent call last):
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/bin/velocyto", line 8, in <module>
    sys.exit(cli())
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/click/core.py", line 764, in __call__
    return self.main(*args, **kwargs)
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/click/core.py", line 717, in main
    rv = self.invoke(ctx)
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/click/core.py", line 1137, in invoke
    return _process_result(sub_ctx.command.invoke(sub_ctx))
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/click/core.py", line 956, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/click/core.py", line 555, in invoke
    return callback(*args, **kwargs)
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/velocyto/commands/run10x.py", line 115, in run10x
    samtools_memory=samtools_memory, dump=dump, loom_numeric_dtype=dtype, verbose=verbose, additional_ca=additional_ca)
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/velocyto/commands/_run.py", line 229, in _run
    results = exincounter.count(bamfile_cellsorted, multimap=multimap)  # NOTE: we would avoid some millions of if statements evaluations if we write two function count and count_with output
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/velocyto/counter.py", line 756, in count
    for r in self.iter_alignments(bamfile, unique=not multimap):
  File "/public-supool/home/wuhaoda/anaconda2/envs/Grim3.6.8/lib/python3.6/site-packages/velocyto/counter.py", line 251, in iter_alignments
    fin = pysam.AlignmentFile(bamfile)  # type: pysam.AlignmentFile
  File "pysam/libcalignmentfile.pyx", line 741, in pysam.libcalignmentfile.AlignmentFile.__cinit__
  File "pysam/libcalignmentfile.pyx", line 951, in pysam.libcalignmentfile.AlignmentFile._open
  File "pysam/libchtslib.pyx", line 365, in pysam.libchtslib.HTSFile.check_truncation

[Arinze-BioX]

I can confirm that I tried @bjbouman’s suggestions and it worked for me. Just ensure that the files/folders in the outs folder are those mentioned here: https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/overview

[bjbouman]

Recently I came across the same error. In my case, there were still some temporary files in the “outs” folder from previous unsuccessful runs. After deleting those files and starting with a “clean” outs folder containing only the .bam, .bai files and the folder with the barcodes, features and matrix, velocyto ran successfully.