Thank you everyone for giving me advice on how to proceed with the analysis, and for helping me understand DADA2.
As @Nicholas_Bokulich mentioned, I have already removed the primers using cutadapt, but I did not know that p-trim-left was used to remove primers. So thank you very much for helping me understand that. So given this information, I will perform a run with only the trunc parameter and see how it turns out. I will keep you posted.
I also notice that you are trimming from the 5’ of your forward reads by 131 bp! Is there a specific reason for this?
I had chosen the first paramenters to trim the 5’ at that point, just because I wasn’t sure if I could choose the area in the bar plot where there was only the error bar and no actual bar, so I skipped all of those on the first run, just to test it, which resulted in not so great results. I will go ahead and try it w trimming at 20 or so.
As of today, I tested a few parameters and trimming at the lower end of the 5’ gave me better results (45), but I am still loosing quite a bit on the filtering step. So I was quite confused. Sorry I did not load that file initially, but the code was running. I was about to test more paramenters today, kind of blindly based on what I understood of DADA2, but thank you for all of your advice.
I will go ahead and run use
**--p-trunc-q parameter and then test one with only trimming at the 3’.**
Just to clarify, if if I use
--p-trunc-q parameter, I specify the other parameters (trunc/trim)?
From what I see if the documents for DADA2 --p-trunc-q is used to discard any reads that are less than or equal to the value stated. It does say that default:2. Based on what I had read before on the tutorials it said a quality score of > = 20 is a good quality score to chose as a parameter, is this then the number that I should chose for this parameter?
** After running my code with a trunc-q of 20, and having lost 80% of sequences to filtering, I found [this post (Dada2 denoise - why do I have so many reads filtered out) which had a great explanation of the trim-q value that I should work with, given that the default is 2. I will rerun my code w a smaller value and check in, in a different post under this thread.
Again thank you guys for all your help, I definitely could not get through all this work with out the forum and without all of your advice and help.