I have the situation that, I used universal primers 341F and 806R for V3-V4 region. However, in my taxa Barplots, I have only bacteria but no archaea at all. Any suggestion what would be the reason for that?
@SoilRotifer, Here is the steps I used to train my classifier following [RESCRIPt] , Have I done anything in my followed steps that I only targeted bacteria?
Option 2: Install within QIIME 2 environment
- conda install -c conda-forge -c bioconda -c qiime2 -c defaults xmltodict
- pip install git+https://github.com/bokulich-lab/RESCRIPt.git
qiime rescript get-silva-data --p-version '138' --p-target 'SSURef_NR99' --p-include-species-labels --o-silva-sequences silva-138-ssu-nr99-rna-seqs.qza --o-silva-taxonomy silva-138-ssu-nr99-tax.qza
qiime rescript reverse-transcribe --i-rna-sequences silva-138-ssu-nr99-rna-seqs.qza --o-dna-sequences silva-138-ssu-nr99-seqs.qza
“Culling” low-quality sequences with cull-seqs
time qiime rescript cull-seqs --i-sequences silva-138-ssu-nr99-seqs.qza --p-n-jobs 5 --o-clean-sequences silva-138-ssu-nr99-seqs-cleaned.qza
Filtering sequences by length and taxonomy
time qiime rescript filter-seqs-length-by-taxon --i-sequences silva-138-ssu-nr99-seqs-cleaned.qza --i-taxonomy silva-138-ssu-nr99-tax.qza --p-labels Archaea Bacteria Eukaryota --p-min-lens 900 1200 1400 --o-filtered-seqs silva-138-ssu-nr99-seqs-filt.qza --o-discarded-seqs silva-138-ssu-nr99-seqs-discard.qza
Dereplicating in uniq mode
time qiime rescript dereplicate --i-sequences silva-138-ssu-nr99-seqs-filt.qza --i-taxa silva-138-ssu-nr99-tax.qza --p-rank-handles 'silva' --p-mode 'uniq' --o-dereplicated-sequences silva-138-ssu-nr99-seqs-derep-uniq.qza --o-dereplicated-taxa silva-138-ssu-nr99-tax-derep-uniq.qza
Make amplicon-region specific classifier
qiime feature-classifier extract-reads \
*--i-sequences silva-138-ssu-nr99-seqs-derep-uniq.qza *
*--p-f-primer CCTACGGGAGGCAGCAG *
*--p-r-primer GGACTACHVGGGTWTCTAAT *
*--p-n-jobs 2 *
*--p-read-orientation 'forward' *
--o-reads silva-138-ssu-nr99-seqs-341f -806r .qza
Dereplicate extracted region
time qiime rescript dereplicate --i-sequences silva-138-ssu-nr99-seqs-341 f-806r.qza --i-taxa silva-138-ssu-nr99-tax-derep-uniq.qza --p-rank-handles 'silva' --p-mode 'uniq' --o-dereplicated-sequences silva-138-ssu-nr99-seqs- 341 f-806r-uniq.qza --o-dereplicated-taxa silva-138-ssu-nr99-tax- 341f -806r-derep-uniq.qza
build our amplicon-region specific classifier.
qiime feature
-classifier fit
-classifier-naive
-bayes
--i-reference-reads silva-138-ssu-nr99-seqs-341f-806r-uniq.qza --i-reference-taxonomy silva-138-ssu-nr99-tax-341f-806r-derep-uniq.qza --o-classifier silva-138-ssu-nr99-341f-806r-classifier.qza
Looking forward for your kind guidance
Here is my taxa barplots attached
atra-taxa-bar-plots.qzv (833.5 KB)