# False positive in my samples
**URL:** https://forum.qiime2.org/t/false-positive-in-my-samples/27930
**Category:** User Support
**Tags:** dada2
**Created:** [October 11, 2023, 11:39pm UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930 "2023-10-11T23:39:16Z")
**Posts on this page:** 7
**Page:** 1
### Author:  [@Edoardo\_Scali](https://forum.qiime2.org/u/Edoardo_Scali)
#### Post date: [October 11, 2023, 11:39pm UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/1 "2023-10-11T23:39:16Z")
Dear community,
I have a question relative to the DADA2 denoising that I performed.
I am working with a ITS1 dataset with more than 400 samples.
Observing my table.qzv I noticed that I have a lot of ASVs, but these are not present with good coverage in the column "# of Samples Observed In".
of 9000 ASVs, 99% are present in just 1 sample.
Here is the protocol that I have used
> qiime demux summarize
> --i-data reads.qza
> --o-visualization reads.qzv
TRIM CUTADAPT
> qiime cutadapt trim-paired
> --i-demultiplexed-sequences reads.qza
> --p-adapter-f AYTTAAGCATATCAATAAGCGGAGGCT
> --p-front-f AACTTTYRRCAAYGGATCWCT
> --p-adapter-r AGWGATCCRTTGYYRAAAGTT
> --p-front-r AGCCTCCGCTTATTGATATGCTTAART
> --o-trimmed-sequences reads-trimmed.qza
DEMUX SUMMARIZE
> qiime demux summarize
> --i-data reads-trimmed.qza
> --o-visualization reads-trimmed.qza
> qiime dada2 denoise-paired
> --i-demultiplexed-seqs reads-trimmed.qza
> --p-trunc-len-f 190
> --p-trunc-len-r 140
> --o-representative-sequences dada2-rep-seqs.qza
> --o-table dada2-table.qza
> --o-denoising-stats dada2-stats.qza
I suspect that the denosising caused a lot of false positive here, because I was expecting a higher number of ASVs present in more samples.
---
### Author:  [@jphagen](https://forum.qiime2.org/u/jphagen)
#### Post date: [October 12, 2023, 5:53pm UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/2 "2023-10-12T17:53:16Z")
Hi @Edoardo_Scali,
In this piece of your code:
> [@Edoardo\_Scali](#):
>
> TRIM CUTADAPT
>
> > qiime cutadapt trim-paired
> > --i-demultiplexed-sequences reads.qza
> > --p-adapter-f AYTTAAGCATATCAATAAGCGGAGGCT
> > --p-front-f AACTTTYRRCAAYGGATCWCT
> > --p-adapter-r AGWGATCCRTTGYYRAAAGTT
> > --p-front-r AGCCTCCGCTTATTGATATGCTTAART
> > --o-trimmed-sequences reads-trimmed.qza
I found that your adapters appear to match those found in [this](https://forum.qiime2.org/t/fungal-its-analysis-tutorial/7351#to-trim-or-not-to-trim-4) tutorial. This could be the problem! Are you using the adapters from your sequencer or from this tutorial? You will want to use the ones that are relevant to your study. I am happy to continue to help you work through this!
-Hannah
---
### Author:  [@Edoardo\_Scali](https://forum.qiime2.org/u/Edoardo_Scali)
#### Post date: [October 13, 2023, 12:10am UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/3 "2023-10-13T00:10:40Z")
Hi @jphagen,
The problem is that I do not have the adapter sequences.
I did not performed
The only oinformation I have about the adapter was obtained from Fastqc reprot and is that these are "Universal Illumina Adapters".
Is there any way to get the adapter sequence from my fastq data?
I have the primers that have been used. Can I use this information for get the adapter sequence?
Thank you so much.
---
### Author:  [@lizgehret](https://forum.qiime2.org/u/lizgehret)
#### Post date: [October 13, 2023, 2:19am UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/4 "2023-10-13T02:19:34Z")
---
### Author:  [@Edoardo\_Scali](https://forum.qiime2.org/u/Edoardo_Scali)
#### Post date: [October 14, 2023, 7:39pm UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/5 "2023-10-14T19:39:04Z")
I was able to fix the problem by following the two step instruction that I found in [this discussion](https://forum.qiime2.org/t/cutadapt-trim-paired-option-front-and-adapter/21372)
After proper primer sequences removal, I performed DADA2 denoising and I got a satisfactory coverage in the column "# of Samples Observed In".
Thank you @jphagen for pointing out that primer removal that I performed was inducing the error!
---
### Author:  [@cherman2](https://forum.qiime2.org/u/cherman2)
#### Post date: [October 16, 2023, 4:04pm UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/6 "2023-10-16T16:04:14Z")
---
### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [November 16, 2023, 10:05pm UTC](https://forum.qiime2.org/t/false-positive-in-my-samples/27930/7 "2023-11-16T22:05:08Z")
This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.