Hello！
I’m using the 515F and 907R primer pair for Illumina sequencing and have processed my paired-end data using the following DADA2 pipeline in QIIME 2:
qiime dada2 denoise-paired \
--i-demultiplexed-seqs paired-end-demux.qza \
--p-trunc-len-f 298 \
--p-trunc-len-r 202 \
--p-trim-left-f 25 \
--p-trim-left-r 25 \
--p-n-threads 64 \
--o-table feature-table.qza \
--o-representative-sequences rep-seqs.qza \
--o-denoising-stats denoising-stats.qza
In this process, I removed 25 base pairs from both the forward and reverse reads to account for the barcodes and primers. I also truncated the reverse reads at 202 bp.
Based on these truncation settings, I expected there to be around 100 bp of overlap between the forward and reverse reads. However, I’ve encountered a problem: in some samples, all sequences are filtered out after denoising, even though there should be enough overlap for merging.
Has anyone encountered this issue before? What might be causing the sequences to be discarded in these samples?
Thank you for any help or suggestions!