Analysis with DADA2

[Abul_Bashar]

Thank you very much @timanix for your constructive response.

@timanix

Here I would prefer to remove primers with cutadapt before running Dada2. Setting special parameter to discard all features in which no primers are found is especially useful since one can see if something went wrong immediately by the size of output file. Also, my intuition says that removing primers with cutadapt rather than trimming will results in more uniformed ASVs after Dada2.

okk. I'll try this and play more with the trunc length to see how it comes up.

@timanix

As I already wrote, V3-V4 region varies in length among different bacteria. Also, trimming primers may affect the length of the output.

I wonder, if DADA2 hits sequence with shorter length after merging, what extent it could affect my result. Currently more than 50% hits are shorter than my expected length (430). In a forum discussion, @Mehrbod_Estaki suggested taxonom-based filtering to avoid any weird hits generated from shorter sequences. Do you think, it could be helpful in my case?

@timanix

That's right but V3-V4 is not only one of the biggest regions to work with, but also varies in size between different bacteria.

I have also noticed that some paper mentioned the varying length of V3-V4 region while using the same primers. This made me a bit confusing.

Thanks again!