# too many gaps in alignment with template

**URL:** <https://forum.mothur.org/t/too-many-gaps-in-alignment-with-template/2679>\
**Category:** Commands in mothur\
**Created:** [January 27, 2016, 5:32pm UTC](https://forum.mothur.org/t/too-many-gaps-in-alignment-with-template/2679 "2016-01-27T17:32:31Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![afioredonno](https://avatars.discourse-cdn.com/v4/letter/a/f475e1/32.png) [@afioredonno](https://forum.mothur.org/u/afioredonno)\
**Post date:** [January 27, 2016, 5:32pm UTC](https://forum.mothur.org/t/too-many-gaps-in-alignment-with-template/2679/1 "2016-01-27T17:32:31Z")

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Dear Patrick, dear mothur users,  
I have two questions, one related to Illumina Miseq read quality (paired-end reads) and one to the way mothur aligns.

1. I noticed that there are many quite large deletions (often of 10 nucleotides) in a very conserved region after the V4 of the 18S (I’m working with a group of protists). Did someone else oberve the same pattern? Was it signaled already?

2. I use mothur to align my reads according to a template. The template doesn’t have any gaps in this region and I used a high penalty for opening gaps (gapopen=-5).  
In the alignment, I noticed that most often the deletions result in several gaps, like this:

T G A A G T A A T A T G A T T G A T A G G G  
T G A A G \_ \_ \_ \_ \_ \_ \_ \_ \_ \_ \_ A T A G G G -\> one deletion of 11 nucleotides, should be one gap  
T \_ \_ \_ G \_ A \_ \_ \_A \_ G \_ \_ \_ \_ A T A G G G -\> the flanking region before the gap is “spread” into the gap, resulting in 5 gaps!

Since the number of gaps can make a difference in the distance between otherwise identical (or closely related) sequences (option “calc” with the default “onegap” in dist.seqs), adding artificial gaps will increase the number of final OTUs. I don’t think that using the “nogap” option would be a solution, since there are other parts of the alignment where gaps have acutally a meaning (difference in lenghts in the variable helices).  
Why does mothur tend to create that many gaps? Is there any option to correct this?  
Waiting for your answer,  
Anna Maria

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**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [January 28, 2016, 1:36pm UTC](https://forum.mothur.org/t/too-many-gaps-in-alignment-with-template/2679/2 "2016-01-28T13:36:05Z")

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I’m not 100% on what you’re asking - a couple things that I think will help. (1) the extra gaps in the SILVA reference are largely structural and really don’t matter. (2) after aligning and screening the sequences, you should run filter.seqs(trump=., vertical=T), which will remove a lot of the structural gaps. (3) in dist.seqs if the two sequences you are comparing have gaps in the same positions, those positions are ignored. The punchline is that I don’t think what you’re worried about is an issue.

Pat