Feature IDs missing from taxonomy

User Support
1

Hi,

I have a quick question. I have previously trained a classifier and assigned taxonomy using the UNITE database for the ITS2 region, and everything was fine. Today, I attempted to create the new classifier (downloaded files here) and used the "sh_qiime_release_16.10.2022.tgz" as well as the " sh_qiime_release_29.11.2022.tgz" to train the classifier, and in both instances, I received the error below.

Please note I used the 29.11.2022 version first, but since I got an error, I decided to try it again with a slightly older version.

I am unsure how to proceed as I have never had this issue. I see that someone else had this issue (here), but I did not change the feature IDs on my table. This post suggested that I used the wrong file (here), but used the same rep-seq I used to create the taxonomy file. Since I've never had this issue before, I do not know how to proceed or how to change or modify the table if I need to. Can you please help?

In case you need this information, my data is composed of 7 libraries (processed intently up to DADA2, and DADA2 files were then merged to create the final files for analysis, as I had previously done).

Here is the script I am currently running: from the classifier to the taxonomy barplot

#Import unite data-------------------------------------------------------------------------------------------------------------------
qiime tools import \
--type FeatureData[Sequence] \
--input-path UNITEclassifier/Classifier/UNITE/developer/sh_refs_qiime_ver9_dynamic_16.10.2022_dev.fasta \
--output-path UNITEclassifier/Classifier/UNITE/developer/unite-seqs-ver9-dynamic-16.10.2022.qza 

#import taxonomy file-----------------------------------------------------------------------------------------------------------
qiime tools import \
--type FeatureData[Taxonomy] \
--input-path UNITEclassifier/Classifier/UNITE/developer/sh_taxonomy_qiime_ver9_dynamic_16.10.2022_dev.txt \
--output-path UNITEclassifier/Classifier/UNITE/developer/unite-ver9-tax-16.10.2022.qza \
--input-format HeaderlessTSVTaxonomyFormat

#Fit the classifier---------------------------------------------------------------------------------------------------------------------
mkdir UNITEclassifier/Classifier

qiime feature-classifier fit-classifier-naive-bayes \
--i-reference-reads UNITEclassifier/Classifier/UNITE/developer/unite-seqs-ver9-dynamic-16.10.2022.qza \
--i-reference-taxonomy UNITEclassifier/Classifier/UNITE/developer/unite-ver9-tax-16.10.2022.qza \
--o-classifier UNITEclassifier/Classifier/unite-ver-classifier-16.10.2022.qza

#Create taxonomy files-------------------------------------------------------------------------------------------------------------#P-read orientation of the primer have to be in reverse 
#complement, using cleaned qza files

mkdir UNITEclassifier/Taxonomy

qiime feature-classifier classify-sklearn \
--i-classifier UNITEclassifier/UNITE/Classifier/unite-ver-classifier-29.11.2022.qza \
--i-reads DADA2/Clean/Fungal-RepSeqs-Merged-1-7-Clean-NS.qza \
--p-read-orientation reverse-complement \
--p-n-jobs 30 \
--o-classification Taxonomy/Fungal-Taxonomy-Clean-16.10.2022.qza 

qiime metadata tabulate \
--m-input-file UNITEclassifier/Taxonomy/Fungal-Taxonomy-Clean-16.10.2022.qza 
--o-visualization UNITEclassifier/Taxonomy/Fungal-Taxonomy-Clean-16.10.2022.qzv

#Create a TaxonomyBarplot------------------------------------------------------------------------------------------------------
qiime taxa barplot \
--i-table DADA2/Clean/Fungal-Table-Merged-1-7-Clean-NS1.qza \
--i-taxonomy UNITEclassifier/Taxonomy/Fungal-Taxonomy-Clean-16.10.2022.qza \
--m-metadata-file Metadata/Fungal-Metadata-Clean-Lib1-7.tsv \
--o-visualization UNITEclassifier/Taxonomy/Fungal-Taxa-Barplot-Clean-16.10.2022.qzv

Thank you so much for all your help,

2

Hi and welcome back to the forum.
So far your code looks fine to me, and I guess that the error is not related to the pipeline itself but probably to the input files.

Are there some differences in origin / creation history between files with NS and NS1 in the names?

If it is not an issue please share your error itself in more details.

Best,

3

Hi! Thank you so much! I honestly would not have learned and finished my Master's without this forum, so thank you all :blue_heart:

oh, duh :woman_facepalming:! Idk how I missed this, especially since this was stated in the previous post. I reran the analysis with the correct file, and it all worked.

Sorry about this mess :slight_smile: and thank you a million.