Error during Relative abundance step

Hello, I’m using ampliseq pipeline and have an error during the relative abundance step, so probably a classifier error but I don’t see where could be the issue.

The error :

Error executing process > ‘RelativeAbundanceReducedTaxa (1)’

Caused by:
Process RelativeAbundanceReducedTaxa (1) terminated with an error exit status (1)

Command executed:

##on several taxa level

array=( 2 3 4 5 6 7 )
for i in ${array[@]}
#collapse taxa
qiime taxa collapse --i-table filtered-table.qza --i-taxonomy taxonomy.qza --p-level $i --o-collapsed-table table-$i.qza
#convert to relative abundances
qiime feature-table relative-frequency --i-table table-$i.qza --o-relative-frequency-table relative-table-$i.qza
#export to biom
qiime tools export relative-table-$i.qza --output-dir relative-table-$i
#convert to tab seperated text file
biom convert -i relative-table-$i/feature-table.biom -o rel-table-$i.tsv --to-tsv

Command exit status:

Command output:
Saved FeatureTable[Frequency] to: table-2.qza
Saved FeatureTable[RelativeFrequency] to: relative-table-2.qza

Command error:
Plugin error from taxa:

Requested level of 3 is larger than the maximum level available in taxonomy data (2).

And here is the command used to create the classifier :

singularity exec ampliseq-1.0.0.simg qiime tools import --type FeatureData[Sequence] --input-path silva_132_99_16S.fna --output-path silva_132_99_16S.qza

singularity exec ampliseq-1.0.0.simg qiime feature-classifier extract-reads --i-sequences silva_132_99_16S.qza --p-f-primer AGAGTTTGATCMTGGCTCAG --p-r-primer ATTACCGCGGCTGCTGGC --p-identity 80 --o-reads silva_v1v3_reads.qza

singularity exec ampliseq-1.0.0.simg qiime tools import --input-path consensus_taxonomy_7_levels.txt --output-path consensus_taxonomy_7_levels.qza --type ‘FeatureData[Taxonomy]’ --source-format HeaderlessTSVTaxonomyFormat

singularity exec ampliseq-1.0.0.simg qiime feature-classifier fit-classifier-naive-bayes --i-reference-reads silva_v1v3_reads.qza --i-reference-taxonomy consensus_taxonomy_7_levels.qza --o-classifier v1v3_99_classifier.qza

So do you have an idea where are the problem ?

It looks like your taxonomy.qza might not have any taxa assigned beyond level 2. (phylum, I think?) I’d probably confirm that this is the case (e.g. by qiime tools export-ing that .qza and taking a quick look at the assignments), and then start troubleshooting the classifier if you do indeed have no assignments beyond level 2.

Let me know what you find, and we’ll go from there. In the meantime, do you have any hypotheses about why this might be happening?

Chris :bear: