Missing output files and coregenom files

[mrojas80]

Hi, I am running PIRATE and am getting an issue where the core genome isn't being created and a few of the output files are missing. The run seems to have gone normally so I am not sure what happened. I checked the log file and it reads as follows:

PIRATE input options:

• Input Directory = /home/mrojas80/Pantoea_102022/GFFs
• Output directory = /home/mrojas80/Pantoea_102022/GFFs/PIRATE
• PIRATE will run using 2 cores
• 436 files in input directory.
• PIRATE will be run on 50,60,70,80,90,95,98 amino acid % identity thresholds.
• PIRATE will be run on features annotated as CDS
• Pangenome contruction will use diamond instead of BLAST

---

Standardising and checking input files:

• 436 gff files passed QC and will be analysed by PIRATE - completed in: 105s

---

• creating co-ordinate files - completed in: 24s
• creating genome loci list: - completed in: 4s

---

Extracting pangenome sequences:

• completed in: 275s

---

Constructing pangenome sequences:

Options:

• Creating pangenome on amino acid % identity using DIAMOND.

• Input directory: /home/mrojas80/Pantoea_102022/GFFs/PIRATE

• Output directory: /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations

• Number of input files: 1

• Threshold(s): 50 60 70 80 90 95 98

• MCL inflation value: 1.5

• Homology test cutoff: 0.001

• Loci file contains 1992482 loci from 436 genomes.

• Extracting core loci during cdhit clustering

• Opening pan_sequences

• /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pan_sequences.fasta contains 1948250 sequences.

• Passing 1948251 loci to cd-hit at 100%

• command: "cd-hit -i /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.temp.fasta -o /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.100 -aS 0.9 -c 1 -T 2 -g 1 -n 5 -M 3480 -d 256 >> /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.cdhit_log.txt"

• Passing 1948251 loci to cd-hit at 99.5%

• command: "cd-hit -i /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.temp.fasta -o /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.99.5 -aS 0.9 -c 0.995 -T 2 -g 1 -n 5 -M 3480 -d 256 >> /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.cdhit_log.txt"

• Passing 1948251 loci to cd-hit at 99%

• command: "cd-hit -i /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.temp.fasta -o /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.99 -aS 0.9 -c 0.99 -T 2 -g 1 -n 5 -M 3480 -d 256 >> /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.cdhit_log.txt"

• Passing 1948251 loci to cd-hit at 98.5%

• command: "cd-hit -i /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.temp.fasta -o /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.98.5 -aS 0.9 -c 0.985 -T 2 -g 1 -n 5 -M 3480 -d 256 >> /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.cdhit_log.txt"

• Passing 1948251 loci to cd-hit at 98%

• command: "cd-hit -i /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.temp.fasta -o /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.98 -aS 0.9 -c 0.98 -T 2 -g 1 -n 5 -M 3480 -d 256 >> /home/mrojas80/Pantoea_102022/GFFs/PIRATE/pangenome_iterations/pan_sequences.cdhit_log.txt"

• completed in 1263 secs

• 0 core loci (0%)

• 1948251 non-core loci (100%)

• 306264 representative loci passed to blast.

• running all-vs-all DIAMOND on pan_sequences

• completed in 11803 secs

• running mcl on pan_sequences at 50

• 33947 clusters at 50 % - completed in 307 secs

• running mcl on pan_sequences at 60

• 41426 clusters at 60 % - completed in 17103 secs

• running mcl on pan_sequences at 70

• 51048 clusters at 70 % - completed in 13369 secs

• running mcl on pan_sequences at 80

• 68152 clusters at 80 % - completed in 9695 secs

• running mcl on pan_sequences at 90

• 111427 clusters at 90 % - completed in 5814 secs

• running mcl on pan_sequences at 95

• 170462 clusters at 95 % - completed in 5014 secs

• running mcl on pan_sequences at 98

• 283919 clusters at 98 % - completed in 5853 secs

• reinflating clusters for pan_sequences

• Finished

I found this error in the slurm log (I can add that as well if you need it):

• ERROR: pangenome_construction.pl failed - error logged at /home/mrojas80/Pantoea_102022/GFFs/PIRATE/fail_test.txt

I then checked the fail_test.txt file:

parallel: Warning: $HOME not set. Using /tmp. parallel: Warning: $HOME not set. Using /tmp. parallel: Warning: $HOME not set. Using /tmp. parallel: Warning: $HOME not set. Using /tmp. parallel: Warning: $HOME not set. Using /tmp. parallel: Warning: $HOME not set. Using /tmp.

The above is all that is written in the file. I don't think this would impact the pangenome construction, so I am not sure what went wrong. I do not get the desired core genome fasta file and other output files.

Any assistance would be greatly appreciated.

[SionBayliss]

It looks like one of you environmental variables ($HOME) hasn't been set. This isn't an issue with PIRATE but with your wider environment. A quick google of "$HOME not set." brings up some potential solutions and flags where this has impacted on other software.

How did you install PIRATE and what environment are you using?

All the best, S

[mrojas80]

You were correct it was a dependency issue! After adding a line to add $HOME to my .bashrc I was able to get all of the outputs when I ran on a small number of files (5). I ran it in a conda virtual environment with all of the dependencies and it now appears to work as intended. Thanks for your assistance!

[SionBayliss]

No problem, glad I could help.

S