First thanks a lot for the support to move forward using qiime2. I read so much in similar questions but still can not move forward. I work with v3-v4 region demultiplexed data. When I got worried because dada2 consider up to 70 % of my sequences as chimeric, with dada2 i have between 50 to 70% ide…

Yes, it will be better to trim at position 400/404 to recover most of the reads. And yes, it will drop all the reads that shorter than this value and trim longer reads

Hello! Looks like primers are still in the reads. Could you try to remove primers ith q2-cutadapt first?

But, when I search for the primers using grep i get 0 as output!! grep -c CCTACGGGAGGCAGCAG /mnt/d/Winter_data/Data_repeats/21Nov74-DL027_S27_L001_R1_001.fastq.gz 0 grep -c GGACTACHVGGGTWTCTAAT /mnt/d/Winter_data/Data_repeats/21Nov74-DL027_S27_L001_R1_001.fastq.gz 0 Is this means Primers are al…

Based on your quality plots, primers are still attached to the sequences. I would try to remove then with cutadapt first, discarding any sequences without primers. If output file is too small compared to original, that's mean that there is either an error with a command or primers are indeed already…

What is the correct primer i should be trimming, the illumina primer at the left or the primer on the right? [image]

I think you should use primers from the right side.

done like that qiime cutadapt trim-paired --i-demultiplexed-sequences /mnt/d/16S_WinterData_Files/H_atra/PrimerRemoved/ atra-demux.qza --p-front-f CCTACGGGAGGCAGCAG --p-front-r GGACTACHVGGGTWTCTAAT --p-match-adapter-wildcards --p-match-read-wildcards --p-discard-untrimmed --o-trimmed-s…

[image] Sabrin: done like that qiime cutadapt trim-paired --i-demultiplexed-sequences /mnt/d/16S_WinterData_Files/H_atra/PrimerRemoved/ atra-demux.qza --p-front-f CCTACGGGAGGCAGCAG --p-front-r GGACTACHVGGGTWTCTAAT --p-match-adapter-wildcards --p-match-read-wildcards --p-discard-untrimmed …

Thank you so much. When I have proceeded, i have generated a set of empty files after deblur. I believe my trimming length was wrong demux-joined.qzv (299.8 KB) [image] (qiime2-2022.2) sabdelghany@LAPTOP-DUNNEENC:/mnt/d/16S_WinterData_Files/H_atra$ qiime deblur denoise-16S --i-demultiplexed-seq…

[image] Sabrin: What is that much wrong with 435? so trimming at 435 here means all reads below that are discarded? This makes no sense to me!! You are right! All reads shorter than 435 will be discarded. The same will happen in Dada2 for each of the reads before merging.

So If I want to keep all my reads as i do not have much, **would it be good to trimm at below 2% percentile so below 404 to keep all of them? But if i trimm at 404 it will drop reads shorter and keep reads longer than 404 but cut those longer reads to the 404 length? Is this correct? [image] …

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