My forward and reverse sequences have min and max length of 80 and 150 respectively.
after importing them into qiime2 , I used dada2 plugin for filtering, merging and feature table construction.
When I checked rep-seqs.qzv file, it showed minimum sequence length 80. This is quite unexpected as a minimum length of the reads itself was 80. Are they really merged sequences??
And even if I see them as technical replicates, they should be very few in numbers. So if I filter these sequences on the basis of frequency, they should get eliminated. but I can still see them in my filtered-rep-seqs.qzv.
Is there any way to remove low length sequences? or any other suggestion?
It's hard for us to know the answer to this without seeing some more details about the actual commands you've ran. Especially the stats output of DADA2 which you can visualize and share with us would be very helpful. But, what you are describing is technically possible if there is a complete overlap between your forward and reverse reads. What is the expected overlap and the length of the final merged products?
I guess this again would depend if they are real merged products with full overlap or somehow erroneous products. What were your filtering parameters?
Hi @Nisha ,
Just wanted to check in here to see if you had any resolution to this or are still waiting for an answer.
Your data2 stats looks ok to me, I don't see any real issues there. As for how there are 80 nts reads, I'm guessing you actually had small target fragments of 80nt that fully overlapped so after DADA2 merged them they were still only 80 nt. If DADA2 fails to merge any pairs, it will drop them both, so in this case they were successfully merged. What is your target region/primers used here? Is it unreasonable to think you may have had 80nts targets given your primers?
I have followed all the steps but couldn't resolve the ultimate problem.
341F and 534R pair of primer was used to get V3-amplicon.
I had posted my query on a separate topic, but it has closed now, how to continue that?
Hi @Nisha ,
Sorry to hear you're still struggling with this. Feel free to open a new thread and point to the other threads with some background, or, if it is a continuation of this post here then you can follow-up here as well. We just want to make sure any one thread is continuous on its own without gaps.