Inconsistent output messages from picrust2

[Zoë Williams]

Hello Robyn/Picrust2 team,

I just ran picrust2 today on two different data sets. Both times, the output messages were not as I had anticipated, but I still got the output files that I expected (KO and EC folders). I am wondering if 1) this indicates concern and if I should be re installing picrust2 and/or re-running my data? Or, 2) if it's not unusual for the system to be dynamic and because I got the anticipated output files it is okay?

What I anticipated to get (ie. the output message I have gotten in the past when running picrust2 with these sorts of data sets):

Warning: There was only one file for the function: KO

Maybe that's fine if you used custom traits or there were no sequences matching one of the domains.

Warning: There was only one file for the function: EC

Maybe that's fine if you used custom traits or there were no sequences matching one of the domains.

The output message I got for Run 1 (other text was weirdly mixed in to the KO part): 

Warning: There was only one file for the function: KO51, ASV.1181, ASV.1113, ASVMaybe that's fine if you used custom traits or there were no sequences matching one of the domains.

Warning: There was only one file for the function: EC

Maybe that's fine if you used custom traits or there were no sequences matching one of the domains.

The output message I got for Run 2 (I got EC folder before the KO folder): 

Warning: There was only one file for the function: EC

Maybe that's fine if you used custom traits or there were no sequences matching one of the domains.

Warning: There was only one file for the function: KO

Maybe that's fine if you used custom traits or there were no sequences matching one of the domains.

Thank you so much in advance for your help! 

Zoë

[Robyn Wright]

Hi Zöe,

I agree that does look a bit weird... could you maybe just try running it again and see if you get the same output? It seems weird as that point in the pipeline isn't running anything on multiple threads so I don't really know how anything else could get printed at the same time (unless you were using something like Parallel and were running multiple commands at the same time?). The ordering of the EC/KO output would depend on whether they were first or second in the list of arguments given when you run, although if you haven't added in the order then the default order of the script is EC and then KO. EC could be second if you had specified KOs but hadn't set no pathways (as EC predictions are required for pathway predictions, so they would then be added in after). What are the commands you have used to run PICRUSt2? But ultimately, I think if you got the expected output then I wouldn't be too concerned. 

Robyn

[Zoë Williams]

Hello Robyn,

Sounds good - thanks for your input! I'll try re running it again over the weekend; based on your feedback it seems that the second run I did was actually giving the appropriate output message (EC first, KO second), so my guess is that re running should fix things. 

This was the command that I ran:

picrust2_pipeline.py \
-s /Volumes/PhD_16S_Data_2025/microbiome_pipeline_zw/picrust2/input/genotype_fasta_RARE.fasta \
-i /Volumes/PhD_16S_Data_2025/microbiome_pipeline_zw/picrust2/input/genotype_otu_RARE.tsv \
-o /Volumes/PhD_16S_Data_2025/microbiome_pipeline_zw/picrust2/output_genotype \
-p 1 \
--in_traits EC,KO \
--no_pathways \
--stratified

Thanks so much!

kindest regards,

Zoë 

[Zoë Williams]

Hello Robyn,

I wanted to follow up as I reran the data using the same command.  Interestingly, KO was still generated before EC, which as previously discussed is surprising, but the output table (specifically "pred_metagenome_unstrat.tsv") was identical to the first run. Thus, although the output messages have been changing it doesn't seem to be influencing the output. 

picrust2_pipeline.py \
-s /Volumes/PhD_16S_Data_2025/microbiome_pipeline_zw/picrust2/input/genotype_fasta_RARE.fasta \
-i /Volumes/PhD_16S_Data_2025/microbiome_pipeline_zw/picrust2/input/genotype_otu_RARE.tsv \
-o /Volumes/PhD_16S_Data_2025/microbiome_pipeline_zw/picrust2/output_genotype \
-p 1 \
--in_traits EC,KO \
--no_pathways \
--stratified

Warning: There was only one file for the function: KO

Warning: There was only one file for the function: EC