Each feature was observed in one sample

Hello everyone:
I met a problem when i used dada2 to denise my pair-end illumina sequences (338F-806R,16S V3-V4). I have 40 samples, each sample have 2 fq files, they are forward and reverse sequences, respectively.
I import these sequences as following codes:

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path mainfest.txt --output-path demux.qza --input-format PairedEndFastqManifestPhred33

qiime demux summarize --i-data demux.qza --o-visualization demux.qzv

And then i denoised the sequences as following codes:
time qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trim-left-f 30 --p-trim-left-r 30 --p-trunc-len-f 250 --p-trunc-len-r 250 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats stats.qza

This process completed without error.
However, i found some feature was only observed in one sample. For example, these 3 samples are time-series samples, all ASVs in G_4d sample could not be found in G_2d and G_6d, i mean ASVs in G_4d only present in this sample, it is unusual.
About 5 of 40 samples are unusal, the ASVs in these samples are unique!

There must be something worry with this result, but i can't figure it out. Could anybody help me? Thank you very much~~

Hi @whl,

A couple of things to consider/question:

  1. What is your sequencing depth? Is it the same across all your samples? If you have a feature present at 1/5000 and one sample have 1000 sequences and one sample has 10000, do you except to see the feature in both?

  2. What is the relative abundance of your “missing” features?

  3. Are these technical replicates or a time series? If they’re a timeseries, why do you expect the same feature to be present in every sample?

  4. Do the samples still cluster together in beta diversity (i.e. are within individual more similar than between individual)? Is this expected behavior?


i used dada2 to denoise my pair-end illumina sequences (16S V4 region, primer 515F-806R). I have two samples, but finally i end up with a each feature is only present in a single sample. Like this:

I think barcode or primer have been removed, bacause I have trimed left 30 bp, the code is following:
time qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trim-left-f 30 --p-trim-left-r 30 --p-trunc-len-f 190 --p-trunc-len-r 190 --o-representative-sequences rep-seqs.qza --
o-table table.qza --o-denoising-stats stats.qza
Does anybody know how to solve this?
Thanks very much!!

Hi @whl,

For your second set of samples, you have tried using cutadapt to trim your primers rather than trimming the the bases? This will give you a more accurate way to make sure your primer sequences are excluded.


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