I am using qiime2-2024.5 version for my analysis containing V3-V4 region of 16S rRNA. i have generated taxonomic profile of my data in two ways:
- using a classifier for V3-V4 region which was previously trained using 2023.5 version. I have used 2023.5 version only at 'qiime feature-classifier classify-sklearn' step. I am not getting any file compatibility issue. Is it right to analyse the data on 2024.5 version while the taxonomy is assigned using 2023.5 version? Is it justiable in paper?
- To avoid the above problem i tried training my classifier again using 2024.5 version using following commands:
qiime rescript get-silva-data
--p-version '138.1'
--p-target 'SSURef_NR99'
--o-silva-sequences silva-138.1-ssu-nr99-rna-seqs.qza
--o-silva-taxonomy silva-138.1-ssu-nr99-tax.qza
qiime rescript reverse-transcribe
--i-rna-sequences silva-138.1-ssu-nr99-rna-seqs.qza
--o-dna-sequences silva-138.1-ssu-nr99-seqs.qza
qiime feature-classifier extract-reads
--i-sequences silva-138.1-ssu-nr99-seqs.qza
--p-f-primer CTACGGGNGGCWGCAG
--p-r-primer GGACTACNNGGGTATCTAAT
--o-reads silva-138.1_V3-V4_16S-seqs.qza
--verbose
qiime rescript dereplicate
--i-sequences silva-138.1_V3-V4_16S-seqs.qza
--i-taxa silva-138.1-ssu-nr99-tax.qza
--p-mode 'uniq'
--o-dereplicated-sequences silva-138.1_V3-V4_16S-derep-seqs.qza
--o-dereplicated-taxa silva-138.1-_V3-V4_16S-derep-tax.qza
qiime metadata tabulate --m-input-file silva-138.1-_V3-V4_16S-derep-tax.qza --o-visualization taxonomy_summary.qzv
qiime metadata tabulate --m-input-file silva-138.1_V3-V4_16S-derep-seqs.qza --o-visualization seqs_summary.qzv
########The sequences and taxonomy here showed d__Eukaryota members, therefore i removed them from the tsv files using python code and using the cleaned sequences nad taxonomy files, i trained classifier on 2024.5 version of qiime2 using following commands:
qiime feature-classifier fit-classifier-naive-bayes --i-reference-reads qiime_sequences.qza --i-reference-taxonomy qiime_taxonomy.qza --o-classifier silva-138.1-16S-nr99-V3_V4-classifier-HS.qza
i got the classifier using above command but when i am using :
"qiime rescript evaluate-fit-classifier --i-sequences qiime_sequences.qza --i-taxonomy qiime_taxonomy.qza --o-classifier silva-138.1-evaluation-16S-nr99-V3_V4-classifier-HS.qza --o-observed-taxonomy silva-138.1-predicted-V3_V4-taxonomy-HS.qza --o-evaluation silva-138.1-V3_V4-taxonomy-fit-classifier-evaluation-HS.qza" ,
it shows following error: 'Plugin error from rescript: Passing a set as an indexer is not supported. Use a list instead.'
The classifier that i trained using 'qiime feature-classifier fit-classifier-naive-bayes' on qiime2-2024.5 is not assigning the same set of sequences to family or genus level while my other classifier which was trained on 2023.5 version is successfully assigning the sequences upto genus level. both the classifiers are trained using SILVA 138.1 datasets.
what is the potential cause of this problem and How can i resolve it?
Also, if i proceed with my taxonomy classified via 2023.5 classifier the no. of features in around 800 while with newly trained classifier using 2024.5 the feature count is around 400 only. Please help me, I am really stuck with this!!!!!!!!!!