Dada2 merging doubt about astrobiomike.github.io

[alanxelena]

Well, that certainly worked

| dada2_input | filtered | dada_f | dada_r | merged | nochim | final_perc_reads_retained
S131 | 26405 | 26389 | 23327 | 24218 | 14838 | 11597 | 44
S187 | 12521 | 12508 | 8537 | 9181 | 3994 | 3890 | 31

That is the best output I could produce by setting truncLen =c(250,244)
It seems that I should think before writing, or maybe writing helped me think...
Anyways, it seems that my overlapping region is only 36 bp long, so I can get rid of 6 nucleotides or so. Bad experimental design I guess.

Thank you for your help!

[AstrobioMike]

Hey there, @alanxelena :)

Thanks for the kind words!

If it is 2x100 sequencing, it might be that they are too short to overlap successfully. Do you know the numerical positions of the primers? Like 515F to 816R, but whatever they are for your case? That will help figure out if the 2x100 is long enough to overlap (and then if maybe the merging-step minimum overlap parameter is the problem). If they are too short to merge, then processing just the forward reads is still an option

[alanxelena]

Hi Mike!

Thank you for your reply! I was thinking about other experiment and mistyped the sequencing conditions, it is actually a 2x250, sorry, my bad. The primers I have been using are the universal ones 341F and 785R which should amplify a product of 464 bp. I will try only processing the forward reads anyhow and let you know how it goes.
Thank you once again!

[AstrobioMike]

Great! That’s rubber ducky programming! Haha :)

https://en.m.wikipedia.org/wiki/Rubber_duck_debugging