I recently got 16S data from Mr DNA which I'm trying to use in Q2 (version 2021.4) for metagenomic analysis. I was able to successfully import my data into Q2 and removed the updated 16S primers (515F/806R) using the following command line:
qiime cutadapt trim-paired
--verbose > cutadapt_log.txt
The resulting reads-cutadapt.qza file (see qzv version attached) doesn't look bad. The median quality scores of base sequences for the forward reads never went below 20, though the 250th base sequence in reverse reads had a median quality score of 11, so I used the following code for denoising:
qiime dada2 denoise-paired
--i-demultiplexed-seqs /home/qiime2/Desktop/16SPostImported_111921/reads-cutadapt.qza --p-trunc-len-f 251 --p-trunc-len-r 249 --o-table /home/qiime2/Desktop/16SPostImported_111921/table.qza
--o-representative-sequences /home/qiime2/Desktop/16SPostImported_111921/rep-seqs_112021.qza --o-denoising-stats /home/qiime2/Desktop/16SPostImported_111921/denoising-stats.qza
However, the resulting table.qza (see .qzv version attached) states that only 56 representative sequences passed the filters, meaning that all of the sequences from a large portion of my samples did not pass the filters.
Am I missing something? Is the truncation command not correct? Could I have used the wrong primer inputs? Any help would be greatly appreciated.