Troubleshooting transcriptome assembly

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.

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