STAR-Fusion error

[Daniel Spagnolo]

Hello, 

I am getting an error running STAR-Fusion "died with ret 11 No such file or directory at /lab/tools/STAR-Fusion-v1.4.0/PerlLib/Pipeliner.pm line 181."

I did see some similar errors in the Google Group suggesting to either:
1. Check that there is adequate RAM or
2. re-make the base installation. 

STAR-Fusion and STAR was installed by our lab in 2018 with a native installation from gitlab source. 

What is interesting to me is we run a small run of samples every other month or so - every other sample on the run had a successful STAR-Fusion analysis, even those with larger fastq files (these are around 11G). And since then we have done another batch that was 100% successful. This suggests to me that the installation is functioning and our analyses have adequate RAM. Analysis is done via HPC. 

The full stack trace is below:

CMD: mkdir -p /lab/projects/rna/1171818/star_fusion
CMD: mkdir -p /lab/projects/rna/1171818/star_fusion/_starF_checkpoints
CMD: mkdir -p /lab/projects/rna/1171818/star_fusion/star-fusion.preliminary
* Running CMD: /lab/tools/STAR-2.6.0c/source/STAR --genomeDir /lab/common/hg19/STAR-Fusion/GRCh37_v19_CTAT_lib_Feb092018/ctat_genome_lib_build_dir/ref_genome.fa.star.idx  --outReadsUnmapped None  --chimSegmentMin 12  --chimJunctionOverhangMin 12  --alignSJDBoverhangMin 10  --alignMatesGapMax 100000  --alignIntronMax 100000  --alignSJstitchMismatchNmax 5 -1 5 5  --runThreadN 16 --outSAMstrandField intronMotif  --outSAMunmapped Within  --outSAMtype BAM Unsorted  --readFilesIn /lab/projects/rna/1171818/MAP23-00013_5622G1_reads1.fastq /lab/projects/MAP23-00013_5622G1_reads2.fastq  --outSAMattrRGline ID:GRPundef  --chimMultimapScoreRange 10 --chimMultimapNmax 10 --chimNonchimScoreDropMin 10  --peOverlapNbasesMin 12 --peOverlapMMp 0.1  --genomeLoad NoSharedMemory  --twopassMode Basic
Error, cmd: /lab/tools/STAR-2.6.0c/source/STAR --genomeDir /lab/common/mgp/hg19/STAR-Fusion/GRCh37_v19_CTAT_lib_Feb092018/ctat_genome_lib_build_dir/ref_genome.fa.star.idx  --outReadsUnmapped None  --chimSegmentMin 12  --chimJunctionOverhangMin 12  --alignSJDBoverhangMin 10  --alignMatesGapMax 100000  --alignIntronMax 100000  --alignSJstitchMismatchNmax 5 -1 5 5  --runThreadN 16 --outSAMstrandField intronMotif  --outSAMunmapped Within  --outSAMtype BAM Unsorted  --readFilesIn /lab/projects/rna/1171818/MAP23-00013_5622G1_reads1.fastq /lab/projects/rna/1171818/MAP23-00013_5622G1_reads2.fastq  --outSAMattrRGline ID:GRPundef  --chimMultimapScoreRange 10 --chimMultimapNmax 10 --chimNonchimScoreDropMin 10  --peOverlapNbasesMin 12 --peOverlapMMp 0.1  --genomeLoad NoSharedMemory  --twopassMode Basic  died with ret 11 No such file or directory at /lab/tools/STAR-Fusion-v1.4.0/PerlLib/Pipeliner.pm line 181.
        Pipeliner::run(Pipeliner=HASH(0x1b1077c0)) called at /lab/tools/STAR-Fusion-v1.4.0/STAR-Fusion line 893
        main::run_STAR(Pipeliner=HASH(0x1b1077c0), "/lab/projects/rna/1171818/MAP23-00013_5622G1_re"..., "/lab/projects/rna/1171818/MAP23-00013_5622G1_re"..., "std", "") called at /lab/tools/STAR-Fusion-v1.4.0/STAR-Fusion line 519

Seems like this is failing around a 2nd pass of the  mapping? Due to this from stdout:

Start job date: Tue Jan 24 16:36:19 EST 2023
Jan 24 16:36:33 ..... started STAR run
Jan 24 16:36:33 ..... loading genome
Jan 24 16:36:43 ..... started 1st pass mapping
Jan 24 16:47:50 ..... finished 1st pass mapping
Jan 24 16:47:52 ..... inserting junctions into the genome indices
Jan 24 16:49:25 ..... started mapping
End job date: Tue Jan 24 16:58:28 EST 2023

and from Log.progress.out:
Jan 26 12:25:43 Started 1st pass mapping

... {11 rows of mapping} ...
Jan 26 12:36:46 Finished 1st pass mapping"
... {7 rows of mapping} ...

There is a completed _STARpass1 directory. 

Thanks, and I really appreciate any help or direction!

Dan

[Brian Haas]

Hi Dan,

This is usually an out of memory issue.  Do you have ~50G of RAM available on the machine you're running this on?

best,

~b

[Daniel Spagnolo]

Hi Brian, 

That was my worry - I'm checking with our HPC engineer now. 

-Dan

[Daniel Spagnolo]

Hi Brian, 

I got the following info from our HPC team:
The 2 master nodes are on dedicated hardware each with 246.75 Gb of RAM and 20 physical processors.  The file-max (max open files limit) is 25389260.

So it seems we would have sufficient RAM, are there any other causes for this type of error I'm receiving? Thanks for any tips you might have!

-Dan 

[Brian Haas]

Hi Dan,

It certainly sounds like memory shouldn't be an issue.

You could try running our latest version of STAR-Fusion. It works off
the same ctat genome lib as before. The default now is to do
single-pass alignment. Maybe that will help.

best,

~brian

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[郝丹]

Hi Brian

I also faced the same problem, I used the version of 1) samtools/1.14   2) R/4.3.0   3) perl/5.30.2   4) star/2.7.10a   5) star-fusion/1.11.0 to run the  STAR-Fusion --genome_lib_dir  GENOME \

           --left_fq CRCnx021xRNA_out_R1.fastq.gz \

             --right_fq CRCnx021xRNA_out_R2.fastq.gz \
             --output_dir ./

The reference is downloaded from the link https://data.broadinstitute.org/Trinity/CTAT_RESOURCE_LIB/__genome_libs_StarFv1.10/GRCh38_gencode_v37_CTAT_lib_Mar012021.plug-n-play.tar.gz.

But I could not get the file of 'star-fusion.fusion_predictions.tsv', but the following files:

And there will have an

Error, cmd: /services/tools/star-fusion/1.11.0/util/STAR-Fusion.map_chimeric_reads_to_genes  --genome_lib_dir /GRCh38_gencode_v37_CTAT_lib_Mar012021.plug-n-play/ctat_genome_lib_build_dir  -J Chimeric.out.junction  -O .//star-fusion.preliminary/star-fusion.junction_breakpts_to_genes.txt --CPU 4  died with ret 256 No such file or directory at /services/tools/star-fusion/1.11.0/PerlLib/Pipeliner.pm line 181.

        Pipeliner::run(Pipeliner=HASH(0xc614f0)) called at /services/tools/star-fusion/1.11.0/STAR-Fusion line 810

what should I do to find the file or directory at /services/tools/star-fusion/1.11.0/PerlLib/Pipeliner.pm line 181 and let it run to get the final files.

I also found some problems with the RAM by others, but I applied for 160gb memory to run. Could you help me with this problem? 

Best wishes,

Dan 

[Brian Haas]

Hi,

That's a peculiar place to break down.

Can you try running this directly?

/services/tools/star-fusion/1.11.0/util/STAR-Fusion.map_chimeric_reads_to_genes  --genome_lib_dir /GRCh38_gencode_v37_CTAT_lib_Mar012021.plug-n-play/ctat_genome_lib_build_dir  -J Chimeric.out.junction  -O .//star-fusion.preliminary/star-fusion.junction_breakpts_to_genes.txt --CPU 4

Also, can you look at the contents of the star-fusion.preliminary/ directory and see if the star-fusion.junction_breakpts_to_genes.txt  file exists there?

Another thing to try is to run the sample data that ships with star-fusion to see if that works. It should run and complete within a few minutes.

best,

~b

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Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

[Brian Haas]

Hi,

So, this error that you're seeing:

IntervalTree.c: loadable library and perl binaries are mismatched (got handshake key 0xdb80080, needed 0xcd00080)

Means there's an installation error.

Try running 'make clean && make' in the base STAR-Fusion installation directory and see if that helps.

If not, then try using our singularity image:

https://github.com/STAR-Fusion/STAR-Fusion/wiki#want-to-use-singularity

best,

~b