hello, I do not understand what is happening, it is the first time that my sequences do not pass the first filter, I would like to understand what may be the reasons for having such a low percentage in the first filter of DADA2, please if someone can give me a hint. I share screenshots of what I’m doing, thank you.
What read length is your sequencing? for example 250bp paired end? When I look at your demultiplexed-sequences-summ.qzv file it indicates your reads are around 271 in the forward and 270 reverse, and you have set
trunc_len_r to those values. In the documents for this, found here. It says:
Position at which forward read sequences should be
truncated due to decrease in quality. This truncates
the 3' end of the of the input sequences, which will
be the bases that were sequenced in the last cycles.
Reads that are shorter than this value will be
discarded. After this parameter is applied there must
still be at least a 12 nucleotide overlap between the
forward and reverse reads. If 0 is provided, no
truncation or length filtering will be performed
So, one issue could be you are throwing away a lot of reads, maybe for a starting point try loosening that threshold and see how you get on?
Thank you very much, I did´t know that reads that were shorter than the value of
trunc_len_r were discarted. The problem is solved! thank you!