I write this here in case someone else has the same doubt that me.
I am working with the amazing q2-sidle, that let to work with short fragments sequences. (The tutorial's link is: Read Preparation — q2-sidle 2020.8 documentation).
My samples are paired-end, demultiplexed by sample but not by region(s). And the sequencing technology is Mi-Seq (Illumina).
Well, in the tutorial suggest that if the user has more than one region to study, the sequences (demultiplexed) should be demultiplexed again by region. To do that, recommend the command
qiime cutadapt trim-paired in which you can eliminate the adapters at the same time you select the region with the choosed primers.
My first question is: Where should I indicate the primers? With the adapters (adapters +primers)?
The specification I have followed are:
q2-sidledocumentation (Read Preparation — q2-sidle 2020.8 documentation).
qiime cutadaptdocumentation (User guide — Cutadapt 3.4 documentation).
But I do not find anything related to primers inside cutadapt documentation. I tried with the adapters sequence and with the primer sequence (separately). However, I have problems with the denoising when I use the primers in cutadapt.
Also, after that, each sequence should be denoise using deblur (Illumina sequences) or dada2 (ION-TORRENT or pyrosequencing 454); right?
And, the protocol of denoising I follow has the next steps (Alternative methods of read-joining in QIIME 2 — QIIME 2 2021.2.0 documentation):
- Joined sequences (I will use deblur and this step is required). Command:
qiime vsearch join-pairs.
- Sequences quality control
- Deblur with
- See feature table obtained with deblur.
Well, when I follow the order (cutadapt + deblur steps), I cannot pass of step 2. And, when I look the plots:
The boxplot obtain after join steps is:
But, after the quality control:
I think I add all the relevant information, I am new with qiime and I am a bit lost yet.
Thank you for your help!