Dear all,
I am currently working with Illumina 2x250bp V4 region amplicon data.
My DADA2 truncation and trimming parameters were the following:
First_truncation:
--p-trim-left-f 10
--p-trim-left-r 10
--p-trunc-len-f 240
--p-trunc-len-r 215 \
second_truncation:
--p-trim-left-f 10
--p-trim-left-r 10
--p-trunc-len-f 240
--p-trunc-len-r 240 \
As a result, First_truncation had 3114 features and second_truncation had 2905 features. So, shorter read produced more feature number which is consistent with the previous forum on this matter.
But my question is on negative control filtering. First_truncation retained 30% of negative control reads whereas second_truncation retained only 20% of negative control reads. These negative controls are blank and they are supposed to have mostly non-real reads. Thus, I thought maybe I should be using second_truncation for the downstream analysis. Would anyone provide an insight into this matter and give a guidance? I would greatly appreciate it. Thank you. I have attached the picture of my interactive quality plot.