# Dada2 losing high amount of reads on denoise step
**URL:** https://forum.qiime2.org/t/dada2-losing-high-amount-of-reads-on-denoise-step/12349
**Category:** Technical Support
**Created:** [November 6, 2019, 10:33am UTC](https://forum.qiime2.org/t/dada2-losing-high-amount-of-reads-on-denoise-step/12349 "2019-11-06T10:33:02Z")
**Posts on this page:** 3
**Page:** 1
### Author:  [@wjschuiten](https://forum.qiime2.org/u/wjschuiten)
#### Post date: [November 6, 2019, 10:33am UTC](https://forum.qiime2.org/t/dada2-losing-high-amount-of-reads-on-denoise-step/12349/1 "2019-11-06T10:33:02Z")
Hey everyone,
I've been struggling with dada2 for the past weeks and while i think i have a basic understanding of the program, i am unable to get dada2 to not filter out \>50% of my reads.
I'm running paired-end 18S illumina reads, the adapters and primers are trimmed off leaving 300 bp paired end reads. The quality plots look as follow:
[[Album] ASV quality plots  ](https://imgur.com/a/7hXHkVb)
I'm using a subset of two samples out of nine to configure dada2 parameters.
I entered the raw reads into dada2 without any pre-filtering, i ran the following command:
qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trunc-len-f 220 --p-trunc-len-r 160 --p-n-threads 4 --o-denoising-stats stats.qza --o-table dada-table.qza --o-representative-sequences rep-seqs.qza --verbose
The first sample had 60k reads, the second 70k reads, after filtering. ~45k of both were left, after denoising ~27k and after merging only 9k. So i decided to try the forward read.
I ran the following command for denoise single:
qiime dada2 denoise-single --i-demultiplexed-seqs paired-end-demux.qza --p-trunc-len 220 --p-n-threads 4 --o-denoising-stats stats.qza --o-table dada-table.qza --o-representative-sequences rep-seqs.qza --verbose
This led to the following stats:
|sample-id|input|filtered|denoised|non-chimeric|
|fun1|261792|222325|154986|123869|
|fun2|282574|235014|172750|144535|
I'm still over 50% of my data during the dada2 step it seems denoising is the biggest factor in the forward read run, but i am unable to improve the numbers by tweaking the filter and truncation parameter. I'm starting to feel the problem may be with the data itself, but i have no clue on where the problem may be. Any help on the issue is much appreciated. I have uploaded the files to my dropbox, they can be downloaded with the following link:
[https://www.dropbox.com/s/tjf1uhfkx4jl7ja/FunSampleSubset.rar?dl=0](https://www.dropbox.com/s/tjf1uhfkx4jl7ja/FunSampleSubset.rar?dl=0)
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### Author:  [@Mehrbod\_Estaki](https://forum.qiime2.org/u/Mehrbod_Estaki)
#### Post date: [November 6, 2019, 10:09pm UTC](https://forum.qiime2.org/t/dada2-losing-high-amount-of-reads-on-denoise-step/12349/2 "2019-11-06T22:09:59Z")
Hi @wjschuiten,
I think your hunch is correct that the problem lies with the data itself. I draw this conclusion because as you mentioned even with single-end reads you are losing about half of your reads. You may be able to increase retention a little bit by truncating the forward reads a bit more say down to 180-220 but I imagine the gain would be minimal. Other options is to relax the maxee parameter (change it to say 4) and increase the # of training reads but you should practice caution with relaxing the maxee, usually the discarded sequences are discarded because they are low in quality. Nevertheless I would say that will be your best bet in increasing retained reads in a significant manner. Hope that helps, keep us posted!
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### Author:  [@wjschuiten](https://forum.qiime2.org/u/wjschuiten)
#### Post date: [November 8, 2019, 8:03am UTC](https://forum.qiime2.org/t/dada2-losing-high-amount-of-reads-on-denoise-step/12349/3 "2019-11-08T08:03:40Z")
Hey @Mehrbod_Estaki
Thanks for your reply, i've done several more runs and it seems trimming the first 30-40 bp's off allows me to retain ~65% of my data at truncation 180-200 which is within acceptable values. Giving dada2 very generous quality arguments (trunc-q 0 and max-ee 20) barely affects the amount of filtered reads. Perhaps there are still some primers attached. I'll proceed with these results, thanks for your thoughts.
Wouter