error in picrust2 plugin outputs

Hi Forum,

I am running the PICRUSt2 plugin on QIIME2-amplicon-2024.2. I followed the tutorial, and everything ran smoothly except the output files for all three predictions (EC metagenome, KO metagenome, and MetaCyc pathway abundance). The outputs seem off (see attached), and I can't pinpoint the exact issue.
Neuer Ordner.zip (7.5 MB)

> >qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path manifest_16S.csv --output-path seqs.qza --input-format PairedEndFastqManifestPhred33
> >qiime cutadapt trim-paired --i-demultiplexed-sequences seqs.qza --p-cores 20 --p-front-f CCTACGGGNGGCWGCAG --p-adapter-f GGATTAGATACCCBDGTAGTC --p-front-r GACTACHVGGGTATCTAATCC --p-adapter-r CTGCWGCCNCCCGTAGG --o-trimmed-sequences trimmed-seqs.qza --verbose > cutadapt_trimming.log
> >qiime dada2 denoise-paired --i-demultiplexed-seqs trimmed-seqs.qza --p-trunc-len-f 260 --p-trunc-len-r 200 --p-chimera-method consensus --p-min-fold-parent-over-abundance 4 --p-n-threads 36 --o-table table-denoise-trimmed-seqs.qza --o-representative-sequences rep-denoise-trimmed-seqs.qza --o-denoising-stats stats-denoise-trimmed-seqs.qza
> >qiime rescript orient-seqs --i-sequences rep-denoise-trimmed-seqs.qza --i-reference-sequences silva-138-99-seqs.qza --o-oriented-seqs oriented-rep-denoise-trimmed-seqs.qza --o-unmatched-seqs unmatched-rep-denoise-trimmed-seqs.qza --p-threads 36
> >qiime feature-table merge-seqs --i-data oriented-rep-denoise-trimmed-seqs.qza --i-data unmatched-rep-denoise-trimmed-seqs.qza --o-merged-data rescript-rep-denoise-trimmed-seqs.qza
> >qiime feature-classifier classify-sklearn --i-reads rescript-rep-denoise-trimmed-seqs.qza --i-classifier silva-138-99-nb-classifier.qza --o-classification rescript-rep-denoise-trimmed-seqs.tax.qza --p-n-jobs 36
> >qiime taxa filter-table --i-table table-denoise-trimmed-seqs.qza --i-taxonomy rescript-rep-denoise-trimmed-seqs.tax.qza --p-exclude Unassigned,d_Eukaryota,d__Archaea --o-filtered-table **taxa-filter-rescript-rep-table-denoise-trimmed-seqs.qza**
> >qiime taxa filter-seqs --i-sequences rescript-rep-denoise-trimmed-seqs.qza --i-taxonomy rescript-rep-denoise-trimmed-seqs.tax.qza --p-exclude Unassigned,d_Eukaryota,d__Archaea --o-filtered-sequences **taxa-filter-rescript-rep-denoise-trimmed-seqs.qza**
##used these two filtered outputs from above for PICRUSt plugn below
> >mkdir q2-picrust2
> >cd q2-picrust2
> >qiime picrust2 full-pipeline --i-table mammal_biom.qza --i-seq mammal_seqs.qza --output-dir q2-picrust2_output --p-placement-tool epa-ng --p-threads 20 --p-hsp-method mp --p-max-nsti 2 –verbose
> >qiime picrust2 full-pipeline --i-table taxa-filter-rescript-rep-table-denoise-trimmed-seqs.qza --i-seq taxa-filter-rescript-rep-denoise-trimmed-seqs.qza --output-dir q2-picrust2_output --p-placement-tool epa-ng --p-threads 20 --p-hsp-method mp --p-max-nsti 2 --verbose
> >qiime feature-table summarize --i-table q2-picrust2_output/pathway_abundance.qza --o-visualization q2-picrust2_output/pathway_abundance.qzv
> >qiime diversity core-metrics --i-table q2-picrust2_output/pathway_abundance.qza --p-sampling-depth 226702 --m-metadata-file 16S-seqs-metadata.tsv --output-dir pathabun_core_metrics_out --p-n-jobs 20
> >qiime tools export --input-path q2-picrust2_output/pathway_abundance.qza --output-path pathabun_exported
> >biom convert -i pathabun_exported/feature-table.biom -o pathabun_exported/feature-table.biom.tsv --to-tsv

Here are the commands I used to generate these outputs. Any help would be greatly appreciated.

Best,
DS

Hi @D_S,
Why do you think the outputs seem off?

Hi,

First, the range between the observed values seems quite large. For instance, in the KO table, it ranges anywhere from 0 to 500,000. Isn't that too wide a range? Additionally, it seems that there are just too many features under the KO (>6000) and EC tables (>1900). My samples are from a mouse model (fecal, stomach, and cecum tissue samples). Is it normal to have such a high range of features in such cases?
This is my first time using PICRUSt, so I am unsure about the output.
Best,
DS

@D_S, these don't jump out as out of the ordinary for me. I'm pinging one the picrust developers to see if they have a different perspective.

cc @Robyn_Wright

1 Like

Hi @D_S,

I am trying to get PICRUST2 plugin working (my current q2 environment = qiime2-amplicon-2024.2) and I see you have had some luck installing it. Would you be able to share the steps you took to make this possible? I saw that the plugin mentioned being compatible with qiime2-2023.2, and so I thought maybe my env was simply incompatible. Any help would be much appreciated! Thanks.