I am having difficulties denoising my paired-end data as the quality of each base drops also in the middle of the reads. I am seeing quite a drop after filtering and merging step. Is this ok or should I only use forward reads? If I only use forward reads then will I be missing some data later in taxonomic assignment step or alternatively, if I leave the reverse reads in the analysis, will I be seeing falsely identified taxa? What are your recommendations based on my results
I am using QIIME2 version 2019.7 installed on my workplace server.
Sequencing length for each read is 251 bp. So our paired-end reads covering the V3-V4 regions using primers 337F (16S_F CCTACGGGNGGCWGCAG) and 805R (16S_R GACTACHVGGGTATCTAATCC) should yield in a sequence with a length 805-337 = 468. Overlapping bases should therefore be 502 (sequencing lengths added up) – 468 (amplicon length)= 34
The command I was using is: denoising-stats.qzv (1.2 MB) visualisatsioon.qzv (295.2 KB)
qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza
I am adding visualization of the quality plot of forward reads and denoising stats. Thanks for your help!