After I imported data to qiime2, I did the clustering(q2-vsearch) at 99.6% identity, exclude singleton, mitochondria and chloroplast. And further statistical analyses were performed with rarefied artifacts(commands are not shown).
Qiime1;
add_qiime_labels.py -i fasta_dir -m example_mapping4.txt -c InputFileName -o combined_fasta
validate_mapping_file.py -m example_mapping4.txt -o validate_mapping_file_output
Qiime2;
qiime tools import \
--input-path Raw_Files/combined_seqs.fna \
--output-path Raw_Files/seqs.qza \
--type 'SampleData[Sequences]'
qiime vsearch dereplicate-sequences \
--i-sequences Raw_Files/seqs.qza \
--o-dereplicated-table Raw_Files/table.qza \
--o-dereplicated-sequences Raw_Files/rep-seqs.qza
qiime vsearch cluster-features-de-novo \
--i-table Raw_Files/table.qza \
--i-sequences Raw_Files/rep-seqs.qza \
--p-perc-identity 0.996 \
--o-clustered-table Raw_Files/table-dn-996.qza \
--o-clustered-sequences Raw_Files/rep-seqs-dn-996.qza
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences Raw_Files/rep-seqs-dn-996.qza \
--o-alignment Raw_Files/aligned-rep-seqs.qza \
--o-masked-alignment Raw_Files/masked-aligned-rep-seqs.qza \
--o-tree Raw_Files/unrooted-tree.qza \
--o-rooted-tree Raw_Files/rooted-tree.qza
#Removing reads that don't appear in at least two samples
qiime feature-table filter-features \
--i-table Raw_Files/table-dn-996.qza \
--p-min-samples 2 \
--o-filtered-table Results_Files/table-dn-996.qza
#Removing the mitochondria and chloroplasts;silva138.1full
qiime taxa filter-table \
--i-table Raw_Files/table-dn-996.qza \
--i-taxonomy Raw_Files/full_taxonomy.qza \
--p-exclude mitochondria,chloroplast \
--o-filtered-table Results_Files/full_table-dn-996_Removed.qza