DADA2 QC and denoising optimisation

[Deni_Ribicic]

Hi Guys,

I need a little help to what I can do to optimise output from DADA2 step.

I have paired-end reads from MiSeq (2x300bp) of V3-V4 16S (341F-805R) with ok amount of reads generated per sample (~29K).

Forward reads seem to be of good quality, while reverse not that great:

After running dada2 with default params + --p-trim-left-f 5 --p-trunc-len-f 280 --p-trim-left-r 5 --p-trunc-len-r 200 most of the reads did not pass the first QC step in most of the samples:

After that I tried to play a bit with trim and trunc parameters, still the same. Then, I tried to increase the --p-max-ee (up to 10) for both forward and reverse, and lowered the --p-trunc-q to 0- nothing much changed.

I am a bit puzzled what is happening here and what else can be done?

I have done the same steps using forward reads alone, as I thought the bad quality of reverse reads might somehow influence this first QC step, but the outcome was fairly similar. Although the forward reads seem to be of very good quality, these also got filtered out on the first QC step.

Any help appreciated.

PS. the DNA obtained and sent for sequencing was of crappy amount and quality as well.

[vheidrich (Vitor Heidrich)]

Hi,

My first guess would be that the issue is being caused by the --p-trunc-len-f 280. Have you tested replacing it by --p-trunc-len-f 0 (no truncation)?

Take a look at the qiime dada2 denoise-paired docs. I am afraid you're losing most of your forward reads by using --p-trunc-len-f 280 because "Reads that are shorter than this value will be discarded".

Hope this helps!

Vitor

[Deni_Ribicic]

I think you're right, how could I miss this!

Even though I have lowered the 280 when playing with --p-trunc-len-f, it seems it was not enough. I can see now that my median is at 270.

So stupid

Thanks!