ERRORs when adding SNVs using SE RNA-Seq data

Hi! I will be very grateful if someone could guide me a bit. I’m trying to add SNVs to a RNA-Seq BAM file aligned using STAR mapper. Here is the command I run 1 week ago: INFO 2017-11-07 11:22:05.779179 starting /storage/003-Third_party_tools/src/bamsurgeon/bin/addsnv.py called with args: /storage/003-Third_party_tools/src/bamsurgeon/bin/addsnv.py -v ../../bamsurgeon_DREAMlike/cosmic/cosmic_covered_SNV_BS.txt -f ../STAR/SRR653897_STAR_Aligned.sorted.bam -r ../../../001-pipelines/data/hs37d5.fa -o mutbam.bam -p 10 --picardjar /storage/003-Third_party_tools/src/picard/picard.jar --mindepth 3 --minmutreads 1 --tagreads --aligner STAR --alignopts STARrefdir:/storage/001-pipelines/data/STAR_index -s 0.1 --ignorepileup --single

BamSurgeon has been running since 2017-11-07 and it has just finished now with a lot of errors, all of them of the same format:

ERROR	2017-11-07 11:22:05.881453	encountered error in mutation spikein: ['1_2938383_2938383_0.178_T']
ERROR	2017-11-07 11:22:05.894334	encountered error in mutation spikein: ['1_2939014_2939014_0.528_A']
ERROR	2017-11-07 11:22:05.894668	encountered error in mutation spikein: ['1_3328295_3328295_0.01_G']
...
...

The following lines show the last lines printed out in the prompt:

INFO	2017-11-20 10:18:42.301486	haplo_6_30672553_30672553	aligning ['addsnv.tmp/haplo_6_30672553_30672553.tmpbam.92626778-76f5-4187-89d2-7de4b89d3cc5.fastq'] with STAR

************************************************************
ERROR	2017-11-20 10:18:42.316819	encountered error in mutation spikein: ['6_30672553_30672553_0.811_G']
Traceback (most recent call last):
  File "/storage/003-Third_party_tools/src/bamsurgeon/bin/addsnv.py", line 275, in makemut
    aligners.remap_bam(args.aligner, tmpoutbamname, args.refFasta, alignopts, mutid=hapstr, paired=(not args.single), picardjar=args.picardjar)
  File "build/bdist.linux-x86_64/egg/bamsurgeon/aligners.py", line 79, in remap_bam
    remap_STAR_bam(bamfn, threads, fastaref, picardjar, options['STARrefdir'], mutid=mutid, paired=paired)
  File "build/bdist.linux-x86_64/egg/bamsurgeon/aligners.py", line 446, in remap_STAR_bam
    p = subprocess.call(sam_cmd)
  File "/usr/lib/python2.7/subprocess.py", line 523, in call
    return Popen(*popenargs, **kwargs).wait()
  File "/usr/lib/python2.7/subprocess.py", line 711, in __init__
    errread, errwrite)
  File "/usr/lib/python2.7/subprocess.py", line 1343, in _execute_child
    raise child_exception
OSError: [Errno 2] No such file or directory
************************************************************
INFO	2017-11-20 10:18:42.318507	no succesful mutations

I will appreciate any help or clue or a way to check what is going on, and hopefully, be able to follow the analysis without restarting from the beginning.

Thank you very much in advance, Cheers.

[adamewing]

Closing, feel free to reopen if there are still unresolved issues.

[adamewing]

Ah, hope that sorts it then. As I said I’d recommend testing on a few sites to ensure you’re getting the results you want before scaling up. Unfortunately the step you’re at (running STAR) is the time-consuming one so there’s not really any checkpoint to start from that would save time. If there’s anything you can do to further parallelise the run that might help with runtime.