minimap2 error while running TrinityFusion

[Simran Arora]

Hi,

 

I am writing to seek assistance with an issue I encountered while running TrinityFusion. The specific error message I received is: 

Error, cannot locate minimap2 database:  /simran/star_run/lib/ctat_genome_lib_build_dir/ref_genome.fa.mm2 at /usr/local/src/TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion line 268.

Any help or suggestions you can provide would be greatly appreciated.

[Brian Haas]

Hi Simran,

Did you run this step to generate the mm2 index?

https://github.com/TrinityCTAT/CTAT-LR-fusion/wiki#configuring-the-ctat-genome-lib-for-ctat-lr-fusion

ctat-LR-fusion -T long_reads.fastq.gz \
               --genome_lib_dir  /path/to/ctat_genome_lib_build_dir \
               --prep_reference --CPU 4

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[Simran Arora]

The command you shared expects a long-read FASTQ file, but I'm dealing with Illumina short-read data. I'm uncertain about how to handle this parameter. Could you please provide instructions or recommend an alternative method?

ctat-LR-fusion -T long_reads.fastq.gz --genome_lib_dir  /path/to/ctat_genome_lib_build_dir --prep_reference --CPU 4

[Simran Arora]

I followed the manual instructions to set up the CTAT Genome Library for use with Minimap2, using the following command:

TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion --prep_reference_only -T ladeda --genome_lib_dir /path/to/ctat_genome_lib_build_dir

However, this command is producing an error, which is detailed below:

 Skipping CMD: /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/ctat-minimap2/ctat-minimap2 -d /home/nipgr/fusion_benchmarking_tools/output/human/trinity/maize/./ctat_genome_lib_build_dir//ref_genome.fa.mm2 /home/nipgr/fusion_benchmarking_tools/output/human/trinity./ctat_genome_lib_build_dir//ref_genome.fa, checkpoint [/home/nipgr/fusion_benchmarking_tools/output/human/trinity/maize/./ctat_genome_lib_build_dir//__mm2_prep_chkpts/mm2_prep_genome.ok] exists.
* Running CMD: /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/ctat-minimap2/misc/paftools.ctat.js gff2bed /home/nipgr/fusion_benchmarking_tools/output/human/trinity./ctat_genome_lib_build_dir//ref_annot.gtf > /home/nipgr/fusion_benchmarking_tools/output/human/trinity/ctat_genome_lib_build_dir//ref_annot.gtf.mm2.splice.bed
sh: /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/ctat-minimap2/misc/paftools.ctat.js: /usr/bin/env/: bad interpreter: Not a directory
Error, cmd: /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/ctat-minimap2/misc/paftools.ctat.js gff2bed /home/nipgr/fusion_benchmarking_tools/output/human/trinity//ctat_genome_lib_build_dir//ref_annot.gtf > /home/nipgr/fusion_benchmarking_tools/output/human/trinity/ctat_genome_lib_build_dir//ref_annot.gtf.mm2.splice.bed died with ret 32256 No such file or directory at /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/PerlLib/Pipeliner.pm line 181.
Pipeliner::run('Pipeliner=HASH(0xbeac48)') called at /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/ctat-LR-fusion line 569
main::prep_minimap2_reference('/home/nipgr/fusion_benchmarking_tools/output/human/trinity/ma...', '/home/nipgr/fusion_benchmarking_tools/output/human/trinity/ma...', '/home/nipgr/fusion_benchmarking_tools/output/human/trinity/ma...', '/home/nipgr/fusion_benchmarking_tools/output/human/trinity/ma...', '/home/nipgr/fusion_benchmarking_tools/output/human/trinity/ma...') called at /home/nipgr/software/TrinityFusion-v0.4.0/CTAT-LR-fusion/ctat-LR-fusion line 252

[Brian Haas]

You can use the --prep_reference_only parameter, and just make an empty file 'fake_long_reads.fastq' and provide that as the input parameter.  This should allow it to build the index.

If you're using the singularity image, mount the genome library without the :ro extension on the -B for running this.

If you're not using the image, then you would need to install 'k8' for use with the minimap2 accessory:

  # K8 installation on linux:
curl -L https://github.com/attractivechaos/k8/releases/download/v0.2.4/k8-0.2.4.tar.bz2 | tar -jxf - && \
    cp k8-0.2.4/k8-`uname -s` $BIN/k8

This is described here under the minimap2 installation instructions:
https://github.com/lh3/minimap2/blob/master/misc/README.md#intro

hope this helps

[Brian Haas]

Hi Simran,

I recently added new ctat genome libs here:
https://data.broadinstitute.org/Trinity/CTAT_RESOURCE_LIB/

that have STAR_v2.7.11a in the name.  These come with the minimap2 index prebuilt as well.  Can you please use this instead? I expect it should solve the issues you've been encountering.

best,

B

On Wed, Nov 1, 2023 at 9:16 AM Simran Arora <sim...@nipgr.ac.in> wrote:

I have followed the steps you outlined previously, but I'm encountering errors with both methods, whether I'm using Singularity or running it locally.

COMMAND USED FOR MOUNTING: singularity exec -e -B /home/sklab/simran/star_run/library/ctat_genome_lib_build_dir/:/ctat_genome_lib trinityfusion.v0.4.0.simg /usr/local/src/TrinityFusion/CTAT-LR-fusion/ctat-LR-fusion --prep_reference_only -T long_read.fastq(fake)--genome_lib_dir /home/sklab/simran/star_run/library/ctat_genome_lib_build_dir/

COMMAND USED FOR RUNNING SAMPLE: singularity exec -e -B ~/simran/star_run/library/ctat_genome_lib_build_dir/:/ctat_genome_lib_build_dir trinityfusion.v0.4.0.simg /usr/local/src/TrinityFusion/TrinityFusion --left_fq ../Input/SRR018266_fastp_R1.fastq --right_fq ../Input/SRR018266_fastp_R2.fastq --output_dir t_out1 --chimeric_junctions ../star_run/out/SRR018266/Chimeric.out.junction --aligned_bam ../star_run/out/SRR018266/Aligned.out.bam --genome_lib_dir ~/simran/star_run/library/ctat_genome_lib_build_dir/

ERROR encountered using singularity:

Running: touch /home/sklab/simran/trinity/t_out1/trinity_fusion-UC_ctat-LR-fusion/FI/chckpts_dir/final.abridged.ok
* Running CMD: /usr/local/src/TrinityFusion/CTAT-LR-fusion/util/merge_mm2fusion_FI.py --mm2_fusions /home/sklab/simran/trinity/t_out1/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI.mm2.fusion_transcripts.breakpoint_info.tsv.w_LR_FFPM  --FI_fusions FI/finspector.FusionInspector.fusions.tsv --output_file /home/sklab/simran/trinity/t_out1/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/mm2_and_FI_fusions_merged.tsv
INFO:__main__:-parsing /home/sklab/simran/trinity/t_out1/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI.mm2.fusion_transcripts.breakpoint_info.tsv.w_LR_FFPM
INFO:__main__:-parsing FI/finspector.FusionInspector.fusions.tsv
INFO:__main__:-merging data frames.
Traceback (most recent call last):
  File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/util/merge_mm2fusion_FI.py", line 44, in <module>
    main()
  File "/usr/local/src/TrinityFusion/CTAT-LR-fusion/util/merge_mm2fusion_FI.py", line 33, in main
    merged_df = pd.merge(mm2_df, FI_df,
  File "/usr/local/lib/python3.8/dist-packages/pandas/core/reshape/merge.py", line 148, in merge
    op = _MergeOperation(
  File "/usr/local/lib/python3.8/dist-packages/pandas/core/reshape/merge.py", line 741, in __init__
    self._maybe_coerce_merge_keys()
  File "/usr/local/lib/python3.8/dist-packages/pandas/core/reshape/merge.py", line 1401, in _maybe_coerce_merge_keys
    raise ValueError(msg)
ValueError: You are trying to merge on object and int64 columns. If you wish to proceed you should use pd.concat
Error, cmd: /usr/local/src/TrinityFusion/CTAT-LR-fusion/util/merge_mm2fusion_FI.py --mm2_fusions /home/sklab/simran/trinity/t_out1/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/LR-FI.mm2.fusion_transcripts.breakpoint_info.tsv.w_LR_FFPM  --FI_fusions FI/finspector.FusionInspector.fusions.tsv --output_file /home/sklab/simran/trinity/t_out1/trinity_fusion-UC_ctat-LR-fusion/fusion_intermediates_dir/mm2_and_FI_fusions_merged.tsv 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(0x557e68005840)) 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 /home/sklab/simran/star_run/library/ctat_genome_lib_build_dir/  --left_fq /home/sklab/simran/trinity/../Input/SRR018266_fastp_R1.fastq  --right_fq /home/sklab/simran/trinity/../Input/SRR018266_fastp_R2.fastq  --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(0x55b0180b27e8)) called at /usr/local/src/TrinityFusion/TrinityFusion line 283