Hi, I am running version qiime2-2021.11 that was conda installed
I have 176 paired-end samples that are illumina processed and are Casava formatted files. I am attempting to look at fungal communities in plant root and soil samples. I am training my own classifier using the UNITE databases and my primers are f/GITS7 and ITS4.
The problem I keep running into is that when I attempt to assign taxonomy all but two of my sample features come back as unidentified. I have been troubleshooting this problem for the last week and have tried many of the fixes recommended to others who have posted about a similar problem.
The code I've been running is this:
IMPORT DATA:
qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path RSD
--input-format CasavaOneEightSingleLanePerSampleDirFmt
--output-path demux-paired-end.qza
DEMULTIPLEX: samples are already demultiplexed and only need to be summarized and viewed
Quality filter and cluster the data into ESVs:
qiime dada2 denoise-paired
--i-demultiplexed-seqs demux-paired-end.qza \
--p-trunc-len-l 265 \
--p-trunc-len-r 233
--o-table table.qza \
--o-representative-sequences rep-seqs.qza
--o-denoising-stats denoising-stats.qza \
--p-n-threads 20 \
SEE DATA DISTRIBUTION:
qiime feature-table summarize \
--i-table table.qza
--o-visualization table.qzv
--m-sample-metadata-file MetadataRGP.tsv
qiime feature-table tabulate-seqs
--i-data rep-seqs.qza
--o-visualization rep-seqs.qzv
qiime metadata tabulate
--m-input-file stats-dada2.qza
--o-visualization stats-dada2.qzv
ASSIGN TAXONOMY:
qiime feature-classifier extract-reads
--i-sequences sh_refs_qiime_ver8_99_s_10.05.2021.qza
--p-f-primer GTGAATCATCGAATCTTTG
--p-r-primer TCCTCCGCTTATTGATATGC
--o-reads ref-seqs.qza
qiime feature-classifier fit-classifier-naive-bayes
--i-reference-reads ref-seqs.qza
--i-reference-taxonomy sh_taxonomy_qiime_ver8_99_s_10.05.2021.qza
--o-classifier classifier.qza
qiime feature-classifier classify-sklearn
--i-classifier classifier.qza
--i-reads rep-seqs-dada2.qza
--o-classification taxonomy.qza
qiime metadata tabulate
--m-input-file taxonomy.qza
--o-visualization taxonomy.qzv
I've tried multiple variations of this with adding steps or other commands such as trying the Classify-consensus-vsearch and ended with the same results. This is my first projects I've used QIIME2 for, I really appreciate any help thank you!