# Dada2 trunc-len 0 for ITS analyses
**URL:** https://forum.qiime2.org/t/dada2-trunc-len-0-for-its-analyses/5691
**Category:** User Support
**Created:** [August 21, 2018, 5:34pm UTC](https://forum.qiime2.org/t/dada2-trunc-len-0-for-its-analyses/5691 "2018-08-21T17:34:50Z")
**Posts on this page:** 4
**Page:** 1
### Author:  [@rahel\_park](https://forum.qiime2.org/u/rahel_park)
#### Post date: [August 21, 2018, 5:34pm UTC](https://forum.qiime2.org/t/dada2-trunc-len-0-for-its-analyses/5691/1 "2018-08-21T17:34:50Z")
Hi,
I am analyzing fungal ITS2 (ITS86F/ITS4 primers, PE300) sequences and I have some questions regarding the truncation of the reads as well as dealing with amplicons of different lengths.
In several sites I have seen that it's advised to put the truncation length at dada2 denoise step to "0".
([https://github.com/Joseph7e/ITS\_metabarcoding\_analyses](https://github.com/Joseph7e/ITS_metabarcoding_analyses)
[DADA2 Pipeline Tutorial (1.16)](https://benjjneb.github.io/dada2/tutorial.html) )
This makes sense as ITS has very variable length depending on the species - using a specific truncation length for all reads will bias the results (as you loose all the shorter amplicons). At the same time when setting the truncation length to 0 I have a lot less sequences that pass the merging step. I tried it as well on data that has been passed through cutadapt.
I found some explanation at this thread [DADA2, truncation lengths and features number](https://forum.qiime2.org/t/dada2-truncation-lengths-and-features-number/1940) - but in the end I still don't know how to proceed with my data so that I could keep all quality reads... Maybe ITSxpress will help (posted another topic on my issues with that tool).
[demux-paired-SB72.qzv](https://cdck-file-uploads-global.s3.dualstack.us-west-2.amazonaws.com/flex002/uploads/qiime21/original/2X/e/e3efca6f0a54ac7f7b4ba6521154b245509733bb.qzv) (270.0 KB)
[trimmed\_sequencesSB72.qzv](https://cdck-file-uploads-global.s3.dualstack.us-west-2.amazonaws.com/flex002/uploads/qiime21/original/2X/4/440baf4780ce0021b32f45ff40f0a5ef7fc32275.qzv) (274.4 KB)
Original data:
qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs demux-paired-end\_SB72.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 270
--p-trunc-len-r 220
--p-n-threads 40
--o-table table\_0SB72.qza
--o-representative-sequences rep-seqs\_SB72.qza
--o-denoising-stats denoising-stats\_truncSB72.qzv
denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 14620 14620 14513 14466
qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs demux-paired-end\_SB72.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 0
--p-trunc-len-r 0
--p-n-threads 40
--o-table table\_0SB72.qza
--o-representative-sequences rep-seqs\_0SB72.qza
--o-denoising-stats denoising-stats\_0SB72.qza
denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 11091 11091 298 298
The cutadapt trimmed data - removed the part that for short amplicons runs into reverse primer
qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs trimmed\_sequences.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 270
--p-trunc-len-r 220
--p-n-threads 40
--o-table table\_trimmedSB72.qza
--o-representative-sequences rep-seqs\_trimmedSB72.qza
--o-denoising-stats denoising-stats\_trimmedSB72.qza
denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 13822 13822 13770 13770
qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs trimmed\_sequences.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 0
--p-trunc-len-r 0
--p-n-threads 40
--o-table table\_0trimmedSB72.qza
--o-representative-sequences rep-seqs\_0trimmedSB72.qza
--o-denoising-stats denoising-stats\_0trimmedSB72.qza
denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 11488 11488 703 703
So in the end I am having trouble with the logical recommendation for ITS not to truncate the reads, but then loosing most of my reads.
Thank you in advance!!!
---
### Author:  [@Nicholas\_Bokulich](https://forum.qiime2.org/u/Nicholas_Bokulich)
#### Post date: [August 22, 2018, 5:36pm UTC](https://forum.qiime2.org/t/dada2-trunc-len-0-for-its-analyses/5691/2 "2018-08-22T17:36:31Z")
> [@rahel\_park](#):
>
> So in the end I am having trouble with the logical recommendation for ITS not to truncate the reads, but then loosing most of my reads.
I think as you noted above, this is because of read-through through the primer at the 3' ends of the reads. So untrimmed reads leave the read-through, which cannot align 😞
Are you sure cutadapt is removing everything? The reverse primers on the forward reads and the forward primers on the reverse reads? From those demux summaries it looks like only a fraction of sequences were actually trimmed, which seems surprising, though with ITS it's always questionable what the expected read length is if you don't know what species are present... 🍄 😭🍄
q2-ITSxpress should take care of the trimming issues 🙂
But otherwise it looks like the trimmed reads are working quite well for you — unless if there's something wrong with that protocol that I'm missing, I'd say run with those if q2-ITSxpress does not fix things for you...
I hope that helps!
---
### Author:  [@rahel\_park](https://forum.qiime2.org/u/rahel_park)
#### Post date: [August 23, 2018, 8:36am UTC](https://forum.qiime2.org/t/dada2-trunc-len-0-for-its-analyses/5691/3 "2018-08-23T08:36:23Z")
Hi,
I'm rather sure that cutadapt removes everything - only fraction was trimmed just because many of the amplicons were very long - so they didn't have the read-through issue.
But finally I agree that the ITSxpress is the way to go 🙂
Thanks!
---
### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [September 23, 2018, 2:36pm UTC](https://forum.qiime2.org/t/dada2-trunc-len-0-for-its-analyses/5691/4 "2018-09-23T14:36:26Z")
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