Taxonomy file has only 30 features

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Hello,

I am analyzing mouse fecal samples. We have sequenced the V3-V4 region of the 16s rRNA and the taxonomic classification has been done based on silva-138-99-tax.qza. The code I used is given below.

1. Importing files into QIIME 2

qiime tools import \
--type 'SampleData[PairedEndSequencesWithQuality]' \
--input-path manifest.tsv \
--output-path demux-paired-end.qza \
--input-format PairedEndFastqManifestPhred33V2.

2. Trimming primer sequences using Cutadapt

qiime cutadapt trim-paired \
--i-demultiplexed-sequences demux-paired-end.qza \
--p-front-f CCTACGGGNGGCWGCAG \
--p-front-r GACTACHVGGGTATCTAATCC \
--p-discard-untrimmed --o-trimmed-sequences demux-pe-trimmed.qza \

–verbose.

3. Denoising using DADA2

qiime dada2 denoise-paired \
--i-demultiplexed-seqs demux-pe-trimmed.qza \
--p-trunc-len-f 278 \
--p-trunc-len-r 119 \
--o-table table.qza \
--o-representative-sequences rep-seqs.qza \
--o-denoising-stats denoising-stats.qza \

4. Assigning Taxonomy

qiime feature-classifier extract-reads \
--i-sequences silva-138-99-seqs.qza \
--p-f-primer CCTACGGGNGGCWGCAG \
--p-r-primer GACTACHVGGGTATCTAATCC \
--o-reads ref-seqs.qza

qiime feature-classifier fit-classifier-naive-bayes \
--i-reference-reads ref-seqs.qza \
--i-reference-taxonomy silva-138-99-tax.qza \
--o-classifier classifier_naive_bayes.qza

qiime feature-classifier classify-sklearn \
--i-classifier classifier_naive_bayes.qza \
--i-reads rep-seqs.qza \
--o-classification taxonomy.qza

I have trained my own classifer based only the reference sequences in the silva database. My taxonomy.qzv file shows only 30 features in it. I am not sure why the number is so low.
taxonomy.qzv (1.2 MB)

2

Hello!
Thank you for providing details regarding the command you run.

Since you are working with V3-V4 region, which is pretty long, my guess is that you need to look into your Dada2 output. Check stats file from the output - how many reads passed the filtering step and how many reads were merged? If you lost most of the reads at merging step, that means that you provided too strict truncation parameters.

Best,
Timur

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