PCoA result differed based on primer trimming methods


I would like to inquire about the primer trimming methods that led to variations in the outcomes.
I have tried two ways to trim the primers: the first is to input demux.qza directly into DADA2 and trim the primers, and the other is to trim with cutadapt before DADA2 denoising. I am using the V1-V2 region of the 16S rRNA region for the analysis.

The problem is that the PCoA plot of unweighted UniFrac distance was different depending on which primer trimming method I used.
When I trimmed the primers using DADA2, there were clear separations between the samples on axis 2 and axis 3. On the other hand, when I trimmed the primers using cutadapt, there were no clear separations.
As for the PCoA plot of weighted UniFrac distance, there were no clear separations between the samples on these axes with either method.

Please inform me of the cause of this issue and provide instructions for resolving any errors in the process.

The following codes were used for the analysis.
QIIME2 version: qiime2-amplicon-2023.9

[Witout cutadapt (primer trimming on DADA2)]

% qiime dada2 denoise-paired \
--i-demultiplexed-seqs demux.qza \ 
--p-trim-left-f 21 \
--p-trim-left-r 20 \
--p-trunc-len-f 300 \
--p-trunc-len-r 236 \
--o-table table.qza  \
--o-representative-sequences rep-seqs.qza \
--o-denoising-stats denoising-stats.qza \

[With cutadapt]

% qiime cutadapt trim-paired \
--i-demultiplexed-sequences demux.qza \
--p-discard-untrimmed \
--o-trimmed-sequences demux_trimmed.qza 

% qiime dada2 denoise-paired \
--i-demultiplexed-seqs demux.qza \ 
--p-trim-left-f 0 \
--p-trim-left-r 0 \
--p-trunc-len-f 279 \
--p-trunc-len-r 216 \
--o-table table_trimmed.qza \
--o-representative-sequences rep-seqs_trimmed.qza \
--o-denoising-stats denoising-stats.qza

The following commands are the same for both methods.

% qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences rep-seqs.qza \
--o-masked-alignment masked-aligned-rep-seqs.qza \
--o-tree unrooted-tree.qza \
--o-rooted-tree rooted-tree.qza \
--o-alignment aligned-rep-seqs.qza

% qiime diversity core-metrics-phylogenetic \
 --i-phylogeny rooted-tree.qza \
 --i-table table.qza \
 --p-sampling-depth 21470 \
 --m-metadata-file metadata_all.txt \
 --output-dir core-metrics-results

% qiime emperor plot \
 --i-pcoa core-metrics-results/unweighted_unifrac_pcoa_results.qza \
 --m-metadata-file metadata_all.txt \
 --o-visualization core-metrics-results/unweighted_unifrac_pcoa_results.qzv

Good morning!

This is a hard question because trimming (with cutadapt or DADA2) happens very early in the process and PCoA plots happen very late. This means you have a lot of internal steps to check for differences.

I would look for differences in the number and length of sequences after trimming.
My working hypothesis is that cutadapt has removed a lot of something leading to less separation in the ordination. I'm looking for what was removed, or to prove to myself that nothing was removed and some other step is causing this issue.

We have a mystery! Keep me posted!

This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.