West Coast Green Crab Experiment Part 93

Troubleshooting transcriptome assembly

2026-07-24

My transcriptome assembly finished running! It was a suspiciously fast run, so I went through the log file to see what happened:

#############################################################################
Finished.  Final Trinity assemblies are written to /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir.Trinity.fasta
#############################################################################


Can't open /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir.Trinity.fasta: No such file or directory at /vortexfs1/home/yaamini.venkataraman/.conda/envs/trinity_env/bin/util/support_scripts/get_Trinity_gene_to_trans_map.pl line 7.
mv: cannot stat 'trinity_out_dir.Trinity.fasta': No such file or directory

So the transcriptome waas created, but then the script couldn’t find the actual output file. Turns out, neither could I?? I saw a temporary transcriptome file, but not the actual transcriptome that was written. I started going through the log again to see if I could figure anything out:

--------------------------------------------------------
-------------------- Chrysalis -------------------------
-- (Contig Clustering & de Bruijn Graph Construction) --
--------------------------------------------------------

inchworm_target: /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/both.fa
bowtie_reads_fa: /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/both.fa
chrysalis_reads_fa: /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/both.fa


#######################################################################
Inchworm file: /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/inchworm.fa detected.
Skipping Inchworm Step, Using Previous Inchworm Assembly
#######################################################################

NO TRINITY I NEEDED A NEW ASSEMBLY BASED ON MY REVISED ASSEMBLY PARAMETERS! Since trinity already found an inchworm file it didn’t make a new assembly. Do I need to delete all previous assembly information? Probably. I cleared the trinity_out_dir folder to ensure that a brand new transcriptome assembly would be created. I then queued my job (job ID 2141130) and hoped a real transcriptome would be created this time. I am so ready to be done with this.

2026-07-30

I ended up with a new error today…joy:

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--------------- Inchworm (K=25, asm) ---------------------

-- (Linear contig construction from k-mers) --

----------------------------------------------

-- Skipping CMD: /vortexfs1/home/yaamini.venkataraman/.conda/envs/trinity_env/bin/Inchworm/bin//inchworm --kmers jellyfish.kmers.25.asm.fa --run_inchworm -K 25 --monitor 1   --num_threads 6  --PARALLEL_IWORM   --min_any_entropy 1.0   -L 25  --no_prune_error_kmers  > /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/inchworm.fa.tmp, checkpoint [.iworm.25.asm.ok] exists.

-- Skipping CMD: mv /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/inchworm.fa.tmp /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/inchworm.fa, checkpoint [.iworm_renamed.25.asm.ok] exists.

Thursday, July 30, 2026: 11:12:49 CMD: touch /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/inchworm.fa.finished

NON_FATAL_EXCEPTION: WARNING, no Inchworm output is detected at: /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir/inchworm.fa at /vortexfs1/home/yaamini.venkataraman/.conda/envs/trinity_env/bin/Trinity line 1843.

Thursday, July 30, 2026: 11:12:49 CMD: /vortexfs1/home/yaamini.venkataraman/.conda/envs/trinity_env/bin/util/support_scripts/get_Trinity_gene_to_trans_map.pl /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir.Trinity.fasta > /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir.Trinity.fasta.gene_trans_map

# No butterfly assemblies to report.

Can't open /scratch/yaamini.venkataraman/wc-green-crab/output/06c-trinity/trinity_out_dir.Trinity.fasta: No such file or directory at /vortexfs1/home/yaamini.venkataraman/.conda/envs/trinity_env/bin/util/support_scripts/get_Trinity_gene_to_trans_map.pl line 7.

mv: cannot stat 'trinity_out_dir.Trinity.fasta': No such file or directory

So inchworm didn’t run and the transcriptome wasn’t actually created. Turns out when I cleared trinity_out_dir, I didn’t do a good enough job. There were still some inchworm checkpoint files from 2025 in the folder that needed to be removed! I straight-up removed the folder and requeued the script. I also added a line to the top of the script removing any output from previous runs:

# Clean up any residual/stale run directories from previous attempts
rm -rf ${OUTPUT_DIR}/trinity_out_dir ${OUTPUT_DIR}/trinity_out_dir.Trinity.fasta

Now to wait and see.

Going forward

  1. Tweak transcriptome assembly parameters to reduce the number of assembly artifacts and total supertranscripts
  2. Annotate transcriptome with EnTAP
  3. Remove contaminant sequences identified by EnTAP
  4. Create count matrix for clean transcriptome
  5. Calculate Ex50 and N50 statistics for clean transcriptome
  6. Repeat analysis with clean transcriptome and fuller annotations in edgeR
  7. Identify temperature- and genotype-specific differentially expressed genes at the end of the experiment
  8. Identify genes influenced by both temperature and time
  9. Determine methods for functional analysis
  10. Additional strand-specific analysis in the supergene region
  11. Examine HOBO data from 2023 experiment
  12. Demographic data analysis for 2023 paper
Written on July 24, 2026