Keeping Unassigned Sequences in the barplot – User Support

Hello,

New user here, still trying to set up a workflow that works…

I got as far as assigning Taxonomy using a pre-trained database (silva138_AB_V4_classifier.qza).
However, there are still some unassigned sequences left. When I want to make a barplot, I get an error because some ‘ Feature IDs found in the table are missing from the taxonomy
I can get rid of them by using this command:

Step 1
qiime feature-table filter-features
–i-table output/table-filtered-seq-clustered.qza
–m-metadata-file output/taxonomy.qza
–o-filtered-table output/id-filtered-table.qza

This solves it to generate a barplot, but I feel like these unassigned sequences are important. I want to include them in my analyses.

How can I synchronise my Fetures table and my Sequence table, without removing unassigned sequences?

My complete workflow (with the error) is below:
(Don’t hesitate to comment on it, as I just got started and can use all the suggestions I can get)

Step 2
(qiime2-2023.2) mac-XXX-XXX:registry myname$ cd ~/Documents/registry
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime tools import
–type ‘SampleData[PairedEndSequencesWithQuality]’
–input-path /Users/myname/Documents/Registry/Raw_data
–input-format CasavaOneEightSingleLanePerSampleDirFmt
–output-path output/demux-paired-end.qza
Imported /Users/myname/Documents/Registry/Raw_data as CasavaOneEightSingleLanePerSampleDirFmt to output/demux-paired-end.qza
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime cutadapt trim-paired
–i-demultiplexed-sequences output/demux-paired-end.qza
–p-front-f GTGYCAGCMGCCGCGGTAA
–p-front-r GGACTACNVGGGTWTCTAAT
–p-match-read-wildcards
–quiet
–o-trimmed-sequences output/primers-removed.qza

Step 3
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime dada2 denoise-paired
–i-demultiplexed-seqs output/primers-removed.qza
–p-trim-left-f 0
–p-trim-left-r 0
–p-trunc-len-f 233
–p-trunc-len-r 229
–o-table output/table.qza
–o-representative-sequences output/rep-seqs.qza
–o-denoising-stats output/denoising-stats.qza
Saved FeatureTable[Frequency] to: output/table.qza
Saved FeatureData[Sequence] to: output/rep-seqs.qza
Saved SampleData[DADA2Stats] to: output/denoising-stats.qza

Step 4
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime feature-table summarize
–i-table output/table.qza
–m-sample-metadata-file metadata.tsv
–o-visualization output/table-summary.qzv
Saved Visualization to: output/table-summary.qzv

Step 5
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime feature-table filter-features
–i-table output/table.qza
–p-min-frequency 1
–o-filtered-table output/table-filtered-seq.qza
Saved FeatureTable[Frequency] to: output/table-filtered-seq.qza

Step 6
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime feature-table summarize
–i-table output/table-filtered-seq.qza
–o-visualization output/table-filtered-seq.qzv
Saved Visualization to: output/table-filtered-seq.qzv

(qiime2-2023.2) mac-XXX-XXX:registry myname$ (specify the lowest number of reads a sample must have to be retained for further analysis)
-bash: specify: command not found

Step 7
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime feature-table filter-samples
–i-table output/table-filtered-seq.qza
–p-min-frequency 2000
–o-filtered-table output/filtered-table-samples.qza
Saved FeatureTable[Frequency] to: output/filtered-table-samples.qza

Step 8
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime feature-table summarize
–i-table output/filtered-table-samples.qza
–o-visualization output/filtered-table-samples-summary.qzv
Saved Visualization to: output/filtered-table-samples-summary.qzv

Step 9
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime vsearch cluster-features-de-novo
–i-table output/filtered-table-samples.qza
–i-sequences output/rep-seqs.qza
–p-perc-identity 0.99
–p-threads 6
–o-clustered-table output/table-filtered-seq-clustered.qza
–o-clustered-sequences output/rep-seqs-clustered.qza
Saved FeatureTable[Frequency] to: output/table-filtered-seq-clustered.qza
Saved FeatureData[Sequence] to: output/rep-seqs-clustered.qza

Step 10
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime vsearch uchime-denovo
–i-table output/table-filtered-seq-clustered.qza
–i-sequences output/rep-seqs-clustered.qza
–o-chimeras output/chimeras.qza
–o-nonchimeras output/rep-seqs-nonchimeric.qza
–o-stats output/chimeras-stats.qza
Saved FeatureData[Sequence] to: output/chimeras.qza
Saved FeatureData[Sequence] to: output/rep-seqs-nonchimeric.qza
Saved UchimeStats to: output/chimeras-stats.qza

Step 11
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime feature-classifier classify-sklearn
–i-classifier Classifier/silva138_AB_V4_classifier.qza
–i-reads output/rep-seqs-nonchimeric.qza
–o-classification output/taxonomy.qza
Saved FeatureData[Taxonomy] to: output/taxonomy.qza

(qiime2-2023.2) mac-XXX-XXX:registry myname$ Phylogenetic Diversity and weighted and unweighted UniFrac.
-bash: Phylogenetic: command not found

Step 12
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime phylogeny align-to-tree-mafft-fasttree
–i-sequences output/rep-seqs-nonchimeric.qza
–o-alignment output/aligned-rep-seqs.qza
–o-masked-alignment output/masked-aligned-rep-seqs.qza
–o-tree output/unrooted-tree.qza
–o-rooted-tree output/rooted-tree.qza
Saved FeatureData[AlignedSequence] to: output/aligned-rep-seqs.qza
Saved FeatureData[AlignedSequence] to: output/masked-aligned-rep-seqs.qza
Saved Phylogeny[Unrooted] to: output/unrooted-tree.qza
Saved Phylogeny[Rooted] to: output/rooted-tree.qza

Step 13
(qiime2-2023.2) mac-XXX-XXX:registry myname$ qiime taxa barplot
–i-table output/table-filtered-seq-clustered.qza
–i-taxonomy output/taxonomy.qza
–m-metadata-file metadata.tsv
–o-visualization output/taxa-barplot.qzv
Plugin error from taxa:
Feature IDs found in the table are missing from the taxonomy: {‘87959caafcd3a9754a7ff216c69f10db’, ’08e542b57c85006f23fc55d4afe2e80e’, ‘8e798cd6222603d9ae5335fabd3109d7’, ‘e02e5e20e6c69df17fba1558706899cf’, ‘8ad99eaeb7cfc8078721880d7e431e54’, ‘a54b3d089a55f8a84e35b8ec1521aabf’, ‘d1345a893834131ca5d77b2e66a5c6af’, ‘9facdd58f96e1f72e4e3165c97ba58ab’}
Debug info has been saved to /var/folders/y1/dw4jwvvs7v76_vgklwnbsw0c0000gp/T/qiime2-q2cli-err-hfzla_06.log

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