Hi everyone!

I'm currently processing paired reads for 16S of V3-V4, forward (341F) and reverse (806R), with DADA2 and I was wondering if someone could help explain or confirm what I'm seeing in my results?

My forward reads are 225 nt and reverse are 222 nt (barcodes and primers are removed from F and R). The sequencing facility says on their website the fragment length is 470 bp for V3-V4.

To calculate overlap, I did (225 + 222) - (470) = -23 which is supposed to mean there's a 23 bp gap between my paired reads. If there's a 23 bp gap from the calculations, then how am I getting such a high % of merging (66-88%) in my denoising stats? Is my logic correct?

My code is:

qiime dada2 denoise-paired

--i-demultiplexed-seqs paired-end-seqs.qza

--p-trunc-len-f 0

--p-trunc-len-r 0

--p-chimera-method consensus

--o-representative-sequences rep-seqs-denoise.qza

--o-table rep_seq_feature_table.qza

--o-denoising-stats denoising-stats.qza

--verbose

And here are the files:

reads_trimmed_summary.qzv (307.4 KB)

denoising-stats.qzv (1.2 MB)

I'm happy I got good merging, but not sure how if I'm going off the calculations. Any help is greatly appreciated! Thanks so much.