Hi, i am quite new for this analysis.
I am running analysis of microbiota for gastric biopsy samples of 250 patients.
I already tried to use the workflow suggested in the Moving Picture tutorial (dada2, denoise etc) using a small trial dataset (8 samples). However, the barplot of taxonomy showed 80% are unnasigned.
So i tried to use the closed reference by vsearch, and I could have less “unassigned” in the taxonomy barplot.
I was performing these 7 steps on the small trial dataset and succeed to get taxonomy barplot analysis. However, i got troubles when i run the dataset contains 250 samples.
So my steps were as follows:
Import paired fastq
Closed reference clustering by vsearch (not plugin one)
vsearch -usearch_global seqs.fna -db ./database/SILVA_138_SSURef_NR99_tax_silva.fasta - strand plus -id 0.99 --blast6out blast_mapping_silva.blast6 --matched seq_mapping_silva.fasta -uc otu_table_mapping_silva.uc
convert uc file into biom table.
import biom table into the featuretable.qza for qiime2 and the seq_mapping_silva.fasta as sequence.qza
Create phylogenetic tree
when i run this , i get error because my sequence is more than 1 million
so i cannot continue analyzing core matrics alpha beta diversity .
Then i tried to assign taxonomy by running
qiime feature-classifier classify-sklearn --i-classifier ./database/silva-138-99-nb-classifier.qza --i-reads seq_mapping_silva_TRIAL.qza --o-classification
It took me several days to assign taxonomy.
Do you have any suggestions for me?