EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem

[EsperanzaDai]

I tried to run the example data provided in the git repo and got the following error. I am using the trinityfusion.v0.4.0.simg.

• Running CMD: /usr/local/bin/STAR --runThreadN 4 --genomeDir /mnt/ctat/ref_genome.fa.star.idx --outSAMtype BAM SortedByCoordinate --twopassMode None --alignSJDBoverhangMin 10 --genomeSuffixLengthMax 10000 --limitBAMsortRAM 47271261705 --alignInsertionFlush Right --alignMatesGapMax 100000 --alignIntronMax 100000 --outSAMattributes All --readFilesIn /mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq --genomeFastaFiles /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --outSAMfilter KeepAllAddedReferences --sjdbGTFfile /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --alignSJstitchMismatchNmax 5 -1 5 5 --scoreGapNoncan -6
  Jun 26 15:45:06 ..... started STAR run
  Jun 26 15:45:06 ..... loading genome

EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem
SOLUTION: please contact STAR author at https://groups.google.com/forum/#!forum/rna-star

Jun 26 15:45:32 ...... FATAL ERROR, exiting
Error, cmd: /usr/local/bin/STAR --runThreadN 4 --genomeDir /mnt/ctat/ref_genome.fa.star.idx --outSAMtype BAM SortedByCoordinate --twopassMode None --alignSJDBoverhangMin 10 --genomeSuffixLengthMax 10000 --limitBAMsortRAM 47271261705 --alignInsertionFlush Right --alignMatesGapMax 100000 --alignIntronMax 100000 --outSAMattributes All --readFilesIn /mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq --genomeFastaFiles /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --outSAMfilter KeepAllAddedReferences --sjdbGTFfile /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --alignSJstitchMismatchNmax 5 -1 5 5 --scoreGapNoncan -6 died with ret 26880 No such file or directory at /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/../PerlLib/Pipeliner.pm line 167.
Pipeliner::run(Pipeliner=HASH(0x55de07ac9d98)) called at /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl line 322
Error: Command '/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl --genome /mnt/ctat/ref_genome.fa --patch /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --max_mate_dist 100000 -G /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --CPU 4 --out_prefix finspector.star --out_dir /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir --reads "/mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq"' returned non-zero exit status 2., exit val: 2
Error, command: [ /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl --genome /mnt/ctat/ref_genome.fa --patch /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --max_mate_dist 100000 -G /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --CPU 4 --out_prefix finspector.star --out_dir /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir --reads "/mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq" ] failed, stack trace: [ st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py, lineno:86
st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:813
st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:1967
]
Traceback (most recent call last):
File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector", line 1967, in
FusionInspector().run()
File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector", line 1541, in run
pipeliner.run()
File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py", line 71, in run
cmd.run(checkpoint_dir)
File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py", line 132, in run
raise RuntimeError(errmsg)
RuntimeError: Error, command: [ /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl --genome /mnt/ctat/ref_genome.fa --patch /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --max_mate_dist 100000 -G /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --CPU 4 --out_prefix finspector.star --out_dir /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir --reads "/mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq" ] failed, stack trace: [ st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py, lineno:86
st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:813
st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:1967
]
Error, cmd: /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector --fusions /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/chimeric_read_candidates.FI_listing --genome_lib_dir /mnt/ctat/ --FI_contigs_fa /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI_targets.fa --FI_contigs_gtf /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI_targets.gtf --left_fq /mnt/procdir/example/reads_1.fq --right_fq /mnt/procdir/example/reads_2.fq died with ret 256 No such file or directory at /usr/local/src/TrinityFusion/CTAT-LR-fusion/PerlLib/Pipeliner.pm line 181.
Pipeliner::run(Pipeliner=HASH(0x55927b3006e0)) called at /usr/local/src/TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion line 540
Error, cmd: /usr/local/src/TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion -T trinity_discasm/discasm.Trinity.fasta --genome_lib_dir /mnt/ctat/ --left_fq /mnt/procdir/example/reads_1.fq --right_fq /mnt/procdir/example/reads_2.fq --CPU 4 --min_J 1 --min_sumJS 2 --min_novel_junction_support 3 -o trinity_fusion-UC_ctat-LR-fusion died with ret 512 No such file or directory at /usr/local/src/TrinityFusion/PerlLib/Pipeliner.pm line 186.
Pipeliner::run(Pipeliner=HASH(0x56430f87e688)) called at /usr/local/src/TrinityFusion/TrinityFusion line 283

[brianjohnhaas]

Hi, I think this somewhat rare issue should be resolved in the latest STAR software version, but that's not included in the current singularity or docker images. I can make a patch for it and see if that helps.... more soon.

…

On Wed, Jun 26, 2024 at 3:51 PM EsperanzaDai ***@***.***> wrote: I tried to run the example data provided in the git repo and got the following error. I am using the trinityfusion.v0.4.0.simg. - Running CMD: /usr/local/bin/STAR --runThreadN 4 --genomeDir /mnt/ctat/ref_genome.fa.star.idx --outSAMtype BAM SortedByCoordinate --twopassMode None --alignSJDBoverhangMin 10 --genomeSuffixLengthMax 10000 --limitBAMsortRAM 47271261705 --alignInsertionFlush Right --alignMatesGapMax 100000 --alignIntronMax 100000 --outSAMattributes All --readFilesIn /mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq --genomeFastaFiles /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --outSAMfilter KeepAllAddedReferences --sjdbGTFfile /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --alignSJstitchMismatchNmax 5 -1 5 5 --scoreGapNoncan -6 Jun 26 15:45:06 ..... started STAR run Jun 26 15:45:06 ..... loading genome EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem SOLUTION: please contact STAR author at https://groups.google.com/forum/#!forum/rna-star Jun 26 15:45:32 ...... FATAL ERROR, exiting Error, cmd: /usr/local/bin/STAR --runThreadN 4 --genomeDir /mnt/ctat/ref_genome.fa.star.idx --outSAMtype BAM SortedByCoordinate --twopassMode None --alignSJDBoverhangMin 10 --genomeSuffixLengthMax 10000 --limitBAMsortRAM 47271261705 --alignInsertionFlush Right --alignMatesGapMax 100000 --alignIntronMax 100000 --outSAMattributes All --readFilesIn /mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq --genomeFastaFiles /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --outSAMfilter KeepAllAddedReferences --sjdbGTFfile /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --alignSJstitchMismatchNmax 5 -1 5 5 --scoreGapNoncan -6 died with ret 26880 No such file or directory at /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/../PerlLib/Pipeliner.pm line 167. Pipeliner::run(Pipeliner=HASH(0x55de07ac9d98)) called at /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl line 322 Error: Command '/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl --genome /mnt/ctat/ref_genome.fa --patch /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --max_mate_dist 100000 -G /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --CPU 4 --out_prefix finspector.star --out_dir /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir --reads "/mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq"' returned non-zero exit status 2., exit val: 2 Error, command: [ /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl --genome /mnt/ctat/ref_genome.fa --patch /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --max_mate_dist 100000 -G /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --CPU 4 --out_prefix finspector.star --out_dir /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir --reads "/mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq" ] failed, stack trace: [ st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py, lineno:86 st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:813 st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:1967 ] Traceback (most recent call last): File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector", line 1967, in FusionInspector().run() File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector", line 1541, in run pipeliner.run() File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py", line 71, in run cmd.run(checkpoint_dir) File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py", line 132, in run raise RuntimeError(errmsg) RuntimeError: Error, command: [ /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/util/run_FI_STAR.pl --genome /mnt/ctat/ref_genome.fa --patch /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.fa --max_mate_dist 100000 -G /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir/finspector.gtf --CPU 4 --out_prefix finspector.star --out_dir /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/FI/fi_workdir --reads "/mnt/procdir/example/reads_1.fq /mnt/procdir/example/reads_2.fq" ] failed, stack trace: [ st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/PyLib/Pipeliner.py, lineno:86 st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:813 st: file:/usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector, lineno:1967 ] Error, cmd: /usr/local/src/TrinityFusion/CTAT-LR-fusion/FusionInspector/FusionInspector --fusions /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/chimeric_read_candidates.FI_listing --genome_lib_dir /mnt/ctat/ --FI_contigs_fa /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI_targets.fa --FI_contigs_gtf /mnt/procdir/example/fusion_results/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI_targets.gtf --left_fq /mnt/procdir/example/reads_1.fq --right_fq /mnt/procdir/example/reads_2.fq died with ret 256 No such file or directory at /usr/local/src/TrinityFusion/CTAT-LR-fusion/PerlLib/Pipeliner.pm line 181. Pipeliner::run(Pipeliner=HASH(0x55927b3006e0)) called at /usr/local/src/TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion line 540 Error, cmd: /usr/local/src/TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion -T trinity_discasm/discasm.Trinity.fasta --genome_lib_dir /mnt/ctat/ --left_fq /mnt/procdir/example/reads_1.fq --right_fq /mnt/procdir/example/reads_2.fq --CPU 4 --min_J 1 --min_sumJS 2 --min_novel_junction_support 3 -o trinity_fusion-UC_ctat-LR-fusion died with ret 512 No such file or directory at /usr/local/src/TrinityFusion/PerlLib/Pipeliner.pm line 186. Pipeliner::run(Pipeliner=HASH(0x56430f87e688)) called at /usr/local/src/TrinityFusion/TrinityFusion line 283 — Reply to this email directly, view it on GitHub <#6>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABZRKX6KGFL6DGNON7PBER3ZJMLSXAVCNFSM6AAAAABJ6RN7S2VHI2DSMVQWIX3LMV43ASLTON2WKOZSGM3TMMJUHA4DQNI> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

-- -- Brian J. Haas The Broad Institute http://broadinstitute.org/~bhaas <http://broad.mit.edu/~bhaas>

[EsperanzaDai]

Thank you so much!

[brianjohnhaas]

just need to do a bit of testing and will be ready shortly

…

On Wed, Jun 26, 2024 at 4:56 PM EsperanzaDai ***@***.***> wrote: Thank you so much! — Reply to this email directly, view it on GitHub <#6 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABZRKX5U25VKCB6A6Q3ZU4TZJMTH5AVCNFSM6AAAAABJ6RN7S2VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDCOJSGYYTGNBUHA> . You are receiving this because you commented.Message ID: ***@***.***>

-- -- Brian J. Haas The Broad Institute http://broadinstitute.org/~bhaas <http://broad.mit.edu/~bhaas>

[brianjohnhaas]

I've updated the version of STAR here to 2.11.b: https://data.broadinstitute.org/Trinity/CTAT_SINGULARITY/TrinityFusion/trinityfusion.v0.4.0-post.simg Hopefully this solves the problem. If it doesn't, we might need to rebuild the reference genome STAR index using that version of STAR. best, Brian

…

On Wed, Jun 26, 2024 at 7:49 PM Brian Haas ***@***.***> wrote: just need to do a bit of testing and will be ready shortly On Wed, Jun 26, 2024 at 4:56 PM EsperanzaDai ***@***.***> wrote: > Thank you so much! > > — > Reply to this email directly, view it on GitHub > <#6 (comment)>, > or unsubscribe > <https://github.com/notifications/unsubscribe-auth/ABZRKX5U25VKCB6A6Q3ZU4TZJMTH5AVCNFSM6AAAAABJ6RN7S2VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDCOJSGYYTGNBUHA> > . > You are receiving this because you commented.Message ID: > ***@***.***> > -- -- Brian J. Haas The Broad Institute http://broadinstitute.org/~bhaas <http://broad.mit.edu/~bhaas>

-- -- Brian J. Haas The Broad Institute http://broadinstitute.org/~bhaas <http://broad.mit.edu/~bhaas>

[EsperanzaDai]

Thank you so much! The new container with the new STAR version works!

[brianjohnhaas]

Fantastic! Thx for the update

…

On Fri, Jun 28, 2024 at 1:37 PM EsperanzaDai ***@***.***> wrote: Thank you so much! The new container with the new STAR version works! — Reply to this email directly, view it on GitHub <#6 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABZRKX7CQEQFSAWNNZBCBM3ZJWNPDAVCNFSM6AAAAABJ6RN7S2VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDCOJXGM3TSOBSHA> . You are receiving this because you commented.Message ID: ***@***.***>

-- -- Brian J. Haas The Broad Institute http://broadinstitute.org/~bhaas <http://broad.mit.edu/~bhaas>