Hi, I have a question regarding how I should configure the nf-core Ampliseq pipeline for 16S metabarcoding analysis targeting the V3–V4 region. I used the Zymo Quick-16S Plus NGS Library Prep Kit (V3–V4) (https://zymoresearch.eu/products/quick-16s-plus-ngs-library-prep-kit-v3-v4-udi?srsltid=AfmBOooKXo-LX85v4ocNVcKSIs2Z9jSEPgg7fvqERGKS1Mv_kLrgZrtz) and sequenced the libraries on an Illumina MiniSeq. The sequencing was performed in *paired-end mode*; however, one limitation of this platform is that it produces reads of 2 × 150 bp. This detail appears to strongly affect the read merging step during the DADA2 stage. I used the default quality-based trimming parameters, and the resulting DADA2 statistics are shown in the file DADA2_stats.tsv. I am analyzing butter cheese (queso mantecoso) samples, and I am also attaching the script that I used. After observing the merging issue, I would like to know how I should proceed with the pipeline—whether it would be more appropriate to analyze the data as single-end (SE) or paired-end (PE) reads. This is particularly important because the analysis will be part of a manuscript that we plan to publish, and this is the first time I have encountered this situation. I would greatly appreciate your guidance on how to proceed. Thank you in advance for your support.

Hi there! Merging issues are typical when using too low read length forthe amplified regions. You seem to have primers 341f and 806r, there I would expect length of about 460bp, without primers ~420bp. Thats too long for PE 150bp. Hence merging is likely impossible. You could attempt two approaches to rescue the failed experimental design: (1) analyse as SE, (2) analyse PE with "--mergepairs_strategy concatenate", that might require also "--skip_dada_addspecies", (3) have a look at both runs and compare, choose one method, keep all results. Report in your manuscript what you have done. In case the reviews complain, tell them what you tried and it might be fine, you have tried to get out of the data what you were able to.

Thank you for your response, Daniel. I will apply it and let you know how it goes.