Why are my quality plots worse after primers removal with CutAdapt?

[fgara]

Dear all,

I have the following two quality plots:

Before CutAdapt:

After CutAdapt:

I don't know why are my quality plots looking worse after primers removal with CutAdapt?

The data is from the V3-V4 region of the 16s gene.
I'm using QIIME2 version 2020.6 and the following cutadapt command:

qiime cutadapt trim-paired
--i-demultiplexed-sequences demux.qza
--p-front-f CCTACGGGNGGCWGCAG
--p-front-r GACTACHVGGGTATCTAATCC
--o-trimmed-sequences demux_cutadapt.qza
--verbose

I'm wondering if I did this wrongly?

Many thanks for your help!

[thermokarst]

Hi @fgara, can you share the QZVs that you produced those screenshots from? It would be really helpful for getting a complete picture of what you have done.

The plots you have shared with us are a random subsample of your reads. If you want to generate something that is 100% comparable, you could set the --p-n parameter to the total number of sequences in the library, rather than the default 10,000.

[fgara]

Hi @thermokarst,

Thank you very much for your kind reply!

Unfortunately I can't share the QZV files, but I think your answer already solved my problem!
I did not realize that the plots were a random subsample of my reads. I will try what you suggested below:

@thermokarst

If you want to generate something that is 100% comparable, you could set the --p-n parameter to the total number of sequences in the library, rather than the default 10,000.

Thank you so much for your generous time and help!