Results different after training feature classifier

Hello everyone!

We ran the analysis against the UNITE ITS database, however the result included the genus Ciliophora, which was unexpected. Despite the fact that the genus Ciliophora exists in the kingdom Fungi, all of the sequences classified as Ciliophora must be considered Alveolata sequences (Kingdom Protista) and we would like to remove them from our analysis.

We removed the Ciliophora genus from both files (just one that matched to the genus) and reran the classifier training as follows, using the taxonomy and fasta sequence from the UNITE ITS database.

qiime tools import
--type 'FeatureData[Sequence]'
--input-path unite_sh_refs_qiime_ver8_97.fasta
--output-path unite_sh_refs_qiime_ver8_97.fasta.qza

qiime tools import
--type 'FeatureData[Taxonomy]' [Uploading
--input-format HeaderlessTSVTaxonomyFormat
--input-path unite_taxonomy_qiime_ver8_97.txt
--output-path unite_taxonomy_qiime_ver8_97.txt.qza

qiime feature-classifier fit-classifier-naive-bayes
--i-reference-reads unite_sh_refs_qiime_ver8_97.fasta.qza
--i-reference-taxonomy unite_taxonomy_qiime_ver8_97.txt.qza
--o-classifier unite-its-97-classifier.qza

However, when compared to the previous version, the new runs turn out to be somewhat different. When comparing the ASV tables from the two runs, I noticed that a few ASVs that were previously matched to another taxonomy were no longer matched to groups in the new run. Example output between the 2 experiment is shown here (phylum barchart

) . Any reason why the results are very different just by removing 1 sequence?

Thanks in advance


Hi @kamalghazali welcome to the forum!

It sounds like your reads are in mixed orientations. See this topic for more details on what is happening, and some troubleshooting steps:

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