Is my DADA2 outputs nomarl?

The DADA2 filter excludes most of the leads.

I am a university student in Japan and a beginner in analysis, so please understand that I am not very good at this.

I am using activated sludge samples for analysis with DADA2.
However, when I use "qiime dada2 denoise-paired", I get the following results. (Photo 1)

Most of the input reads are lost at the time of filtering, and nothing remains at the end.
The forward sequence has a high quality score, but the reverse sequence has an extremely low score. (Photo 2)

The actual code used is shown below.

qiime dada2 denoise-paired \
 --i-demultiplexed-seqs paired-end-demux.qza  \
 --p-trunc-q 0 \
 --p-trim-left-f 30 \
 --p-trim-left-r 30 \
 --p-trunc-len-f 230 \
 --p-trunc-len-r 230 \
 --o-representative-sequences rep-seqs-dada2.qza  \
 --o-table table-dada2.qza   \
 --o-denoising-stats stats-dada2.qza \

Adding "--ptrunc-q 1 " did not change the result.

I have two questions.
(1) What is the cause of the problem?
PCR? NGS? DNA extraction? Others?
(2) Can I get any result from this data?
I would like to get some results, excluding the accuracy of the data.


You might want to take a look on that thread:

The quality of the reverse read is OK. There are a lot of variables that influence it.

To get results try changing --p-min-overlap parameter.


Hi crusher!

Thank you for your reply.

I contacted illmina and they had a similar problem with a product with the same lot number.
I decided to rerun the NGS.


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