# Creating a customized reference alignment for V1-V2

**URL:** <https://forum.mothur.org/t/creating-a-customized-reference-alignment-for-v1-v2/20268>\
**Category:** Commands in mothur\
**Created:** [January 4, 2020, 3:17pm UTC](https://forum.mothur.org/t/creating-a-customized-reference-alignment-for-v1-v2/20268 "2020-01-04T15:17:44Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![Joanna](https://avatars.discourse-cdn.com/v4/letter/j/ce73a5/32.png) [@Joanna](https://forum.mothur.org/u/Joanna)\
**Post date:** [January 4, 2020, 3:17pm UTC](https://forum.mothur.org/t/creating-a-customized-reference-alignment-for-v1-v2/20268/1 "2020-01-04T15:17:44Z")

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Hi,  
I follow the MiSeq\_SOP and [this post](https://forum.mothur.org/t/miseq-sop-pcr-seqs/1413/5) to create my customized db for the V1V2 region by using Silva 132. Everything seems to work well; however, I am not getting such consensus results as in the case of MiSeq\_SOP (see the table at the end of this post, it is the result of summary.seqs). Especially, I am worried about the minimum and maximum value. Does this indicate some issue? Do I have to do some additional steps before aligning it to my sequences?

I did:

1. pcr.seqs(fasta=ecoli.16srrna.fasta, oligos= oligos\_primers.txt)  
oligos\_primers.txt looks as follow  
forward GTTYGATYMTGGCTCAG  
reverse GCWGCCWCCCGTAGGWGT

2. align.seqs(fasta=ecoli.16srrna.pcr.fasta, reference= head\_silva.nr\_v132.align)  
Download the most updated version of [Silva](https://www.mothur.org/wiki/Silva_reference_files) (132) “Full length sequences and taxonomy references”.  
This file is pretty big so use head e.g. 29912 (this is the length of the Silva db included in MiSeq\_SOP).

3. align.seqs(fasta=ecoli.16srrna.pcr.fasta, reference= head\_silva.nr\_v132.align)

4. summary.seqs(fasta=ecoli.16srrna.pcr.align)  
Results: start position is 1046 and end is 6333

5. pcr.seqs(fasta=silva.nr\_v132.align, start=1046, end=6333, keepdots=F, processors=16)

6. rename.file(input=silva.nr\_v132.pcr.align, new=silva132.V12.fasta)

7. summary.seqs(fasta=silva132.V12.fasta)

Minimum: 1 3929 214 0 3 1  
2.5%-tile: 1 5286 273 0 3 5328  
25%-tile: 2 5286 299 0 4 53280  
Median: 2 5286 305 0 5 106560  
75%-tile: 2 5286 317 0 5 159840  
97.5%-tile: 2 5286 388 0 6 207792  
Maximum: 74 5286 1047 5 15 213119  
Mean: 1 5285 310 0 4

# of Seqs: 213119

Many thanks, Joanna

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<div class="post-metadata">

**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 9, 2020, 4:12pm UTC](https://forum.mothur.org/t/creating-a-customized-reference-alignment-for-v1-v2/20268/2 "2020-01-09T16:12:56Z")

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Hi,

You might add a `screen.seqs` step like this…

```auto
screen.seqs(fasta=silva.nr_v132.pcr.align, start=2, end=5286)
rename.file(input=silva.nr_v132.pcr.good.align, new=silva132.V12.fasta)

```

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**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\
**Post date:** [January 19, 2020, 4:13pm UTC](https://forum.mothur.org/t/creating-a-customized-reference-alignment-for-v1-v2/20268/3 "2020-01-19T16:13:03Z")

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