Greetings. I'm new to QIIME2. So there are still a lot of things I need to learn and I appreciate all of helps I can receive.
Currently, I'm conducting a 16s QIIME2 analysis pipeline based on moving picture tutorials to know about the workflow and what does each parameter do in each step.
The data I'm using is from this article (V3-V4 paired end 16s 2x250): The pesticides carbofuran and picloram alter the diversity and abundance of soil microbial communities - PMC
The current problem I am meeting is that:
During DADA2, I noticed that a lot of reads were lost during the merge process. And I have already tried to read on the forum about this matter but to be honest, I'm still lost on this matter.
This is the quality plot after importing:
These are all of my version running DADA2 (I mainly played around with p-trunc-f/r since it seems to be the main reason why I lose a lot of reads):
Ver 1 (Forward: 217; Reverse: 196)
Ver 2 (Forward: 250; Reverse: 200)
Ver 3 (Forward: 250; Reverse: 250)
Ver 4 (Forward: 229; Reverse: 205)
From all of 4 vers, I can only see that all 4 didn't retain that many reads during the merging step, only ver 3 can retain around 20%.
Additionally, I have already read about this problem. The problem seems to stem from not enough overlap regions after truncating. But in ver 3, I basically didn't truncate but I still didn't have a lot of reads retained.
Moreover, I tried to read about the impact of parameters via Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing | Nature Methods and DADA2: Fast and accurate sample inference from amplicon data with single-nucleotide resolution and denoise-paired: Denoise and dereplicate paired-end sequences — QIIME 2 2024.10.1 documentation
