Hello.
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]
#Cutadapt
% qiime cutadapt trim-paired \
--i-demultiplexed-sequences demux.qza \
--p-front-f AGRGTTTGATYMTGGCTCAG \
--p-front-r TGCTGCCTCCCGTAGGAGT \
--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