It’s likely that your input demux file is somehow corrupted, but it’s difficult to assess only on the basis of the qzv visualization that you attached. Do you also encounter errors while running qiime deblur on your sequences?
I am using PGM ion torrent instead Miseq but I have he same problem about the orientation of my sequences in single end. I have in each fastq/sample both orientations. Could you help me with the reorientation? I am not sure about how to do it.
regarding your imported sequences, do you have one file per sample or one for all samples? I have one file per sample and to import like demultiplexed file I used one manifest file. Dada and deblur run without errors, but in my case with wrong results due to my mixed orientations.
I'm not familiar with PGM ion torrent but I've provided the script I'm using to orient & trim. Note that it checks for primers on both ends of joined reads. You mention single-ended reads, so the script may have to be modified to handle that case. Also, you'll probably have to update it with your own primers.V4V5_orient_and_trim_primers_fuzzy.py (8.2 KB)
In my orient & trim step, the resulting R1 & R2 reads won't necessarily be the same length (either within a file or between the two files). Is that the issue? Is there any other way I can validate my demux file?
I ran the following with no issues: qiime deblur denoise-16S --i-demultiplexed-seqs seqMAT56_demux.qza --p-trim-length 220 --p-sample-stats --output-dir deblur --p-jobs-to-start 10
and have attached the output visualizations.
I do not know why you had problems in Dada, but in Deblur the results seem normal. Except for the two first samples, which have low sequnces, the rest lose arond 25-36% of sequences because the size of the fragment. Maybe you can try lower trimming, 200, 180, for example, in order to check how many sequences you recover after denoised filter if you use lower size.
I was able to isolate the issue to my trimming and orienting step. In the rare case of a primer being present in only on read direction (either R1 or R2), that read half will get oriented (and potentially reversed complimented) whereas its pair will not. I’ve since updated my orient script to handle said case.
I’m not sure if this is moot, or if either the QIIME2 or DADA2 developers want to update their error reporting for this. If required I can provide example data to re-create this issue.