Some questions about the parameter choosing in DADA2

Hi team,
I am trying to do the dada2 denoising step after I got the demux.qzv file, but I am not sure how to decide the two parameters –p-trim-left and –p-trunc-len, because I think the low quality scores are not shown closely. And there are some red text shown on the result, e.g. "This position (242) is greater than the minimum sequence length observed during subsampling (125 bases). "
I am not sure what happened and what is the correct way to deal with it. I have attached my results, could you please help me with it? (My data is 16s sequencing data)

Thank you so much for any of your kind help!

demux_81-120.qzv (292.5 KB)

Hi @Claire,
Welcome to the forum!
The message you are seeing is just a warning saying that at that particular position (242) of the default 10,000 subsampled reads, 9996 of them are longer than 242, meaning 4 of your reads were actually shorter than 242. The minimum length of at least one of those reads was 125bp. This is nothing to be worry about on its own, and is quite common.
On the other hand, your quality scores actually look rather artificial. Usually we see much more fluidity with regards to the quality scores decreasing as a function of length. Has there been any type of quality filtering or pre-processing done to these reads already? For DADA2, you’ll want to use non-modified raw reads in order for it to properly develop its own error-model.

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