# DADA2, truncation parameters question

**URL:** https://forum.qiime2.org/t/dada2-truncation-parameters-question/13627
**Category:** User Support
**Created:** [February 14, 2020, 7:04pm UTC](https://forum.qiime2.org/t/dada2-truncation-parameters-question/13627 "2020-02-14T19:04:40Z")
**Posts on this page:** 3
**Page:** 1

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### Author: ![elab626](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/e/f17d59/32.png) [@elab626](https://forum.qiime2.org/u/elab626)
#### Post date: [February 14, 2020, 7:04pm UTC](https://forum.qiime2.org/t/dada2-truncation-parameters-question/13627/1 "2020-02-14T19:04:40Z")

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Hello,

I have a question about truncation parameters for a 2x300 bp paired end run using the V3-V4 region. These results are with the primers removed (341F and 805R). Here is the visualization of demux.qzv after trimming primers using cutadapt.

 ![Picture1](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/2X/b/b6d3cfa310230d57427ff1b6ebe2343a2f31ccf6.png)

My first data point is with reads that have been truncated at --p-trunc-len-f 281 and -p--trunc-len-r 202. Theoretically, these limits should be fine given that the expected amplicon size is around ~460 bp. This should leave enough for the ~20 bp DADA2 overlap requirement. As you can see, not much is lost at merging suggesting that this step is working fine.

 ![image](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/2X/a/a477ca1fb94a583301f5745962cdded889dcbe79.png)

I followed up by truncating a bit deeper, to see if the reads still merge. These parameters were set at --p-trunc-len-f 245 and -p--trunc-len-r 191.

 ![image](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/2X/9/9e50140b86d0523190aa06a774b168680a26e594.png)

Now although I lose a little bit more reads at the merging steps compared to the first truncation, I still retain a good amount. However, if the amplicon size is around ~460 bp, shouldn't I be losing a lot more reads at merging, especially accounting for the ~20bp DADA2 overlap requirement? What could explain retaining such a large amount of reads?

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### Author: ![colinbrislawn](https://forum.qiime2.org/user_avatar/forum.qiime2.org/colinbrislawn/32/6221_2.png) [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [February 14, 2020, 7:35pm UTC](https://forum.qiime2.org/t/dada2-truncation-parameters-question/13627/2 "2020-02-14T19:35:36Z")

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Good afternoon,

Welcome to the forums! :qiime2:

> [@elab626](#):
>
> However, if the amplicon size is around ~460 bp, shouldn’t I be losing a lot more reads at merging, especially accounting for the ~20bp DADA2 overlap requirement?

Your original lengths:  
281 + 202 - 460 = 23 bp overlap expected  
245 + 191 - 460 = -24 bp overlap = 24 bp _ **gap** _ expected!

I'm not sure why your reads are pairing with those shorter lengths! Maybe your region is a little shorter than expected? You could try aligning reads to a reference to test, if you really wanted.

For those two trimming lengths, how does the distribution of read lengths differ? Because if both trimming settings make reads that are ~420 bp long, then we have answered our question and are good to go!

Colin

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### Author: ![system](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/3X/2/1/21af5fe23cb6f4579467c66a9ed94e55274ca7bd.svg) [@system](https://forum.qiime2.org/u/system)
#### Post date: [March 17, 2020, 11:10am UTC](https://forum.qiime2.org/t/dada2-truncation-parameters-question/13627/6 "2020-03-17T11:10:01Z")

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