quality score and where to truncate sequence in DADA2?

Hi @Pikula,

I am not an expert on ITS, but I will try to share my little experience!
First of all, on which region the primers anneal? If ITS1 or ITS2 you can use ITSxpress to extract only the ITS region form the sequences: Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences

I would be a bit skeptical to apply a fixed length threshold across all the sequences given the high length variability you giving. I would be worried to loose many of the good longer ITS amplicons. Especially because, looking at the length of your sequences, if you have a max length of 460bp, you have barely an overlap between R1 and R2, so you may want to be fairly relaxed with the trimming to not loosing it.

I would therefore keep ‘–p-trunc-len-f 0’ and ‘–p-trunc-len-r 0’, this will leave on the default quality trimming applied to all the sequences (you may choose a stricter quality then the default if you like!). If you apply ITSxpress, it should behind the scene perform a quality trimming as well.

But again, this is just my thought, hope it helps.

Luca

2 Likes