# PR2.fasta file not aligned

**URL:** <https://forum.mothur.org/t/pr2-fasta-file-not-aligned/3357>\
**Category:** Commands in mothur\
**Created:** [August 7, 2017, 11:30am UTC](https://forum.mothur.org/t/pr2-fasta-file-not-aligned/3357 "2017-08-07T11:30:14Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![NicoleDames14](https://avatars.discourse-cdn.com/v4/letter/n/ecccb3/32.png) [@NicoleDames14](https://forum.mothur.org/u/NicoleDames14)\
**Post date:** [August 7, 2017, 11:30am UTC](https://forum.mothur.org/t/pr2-fasta-file-not-aligned/3357/1 "2017-08-07T11:30:14Z")

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Hi,

I am attempting align against the template pr2.fast, but only get the following error:

  
mothur \> align.seqs(fasta=18S.unique.fasta, reference=pr2\_gb203\_version\_4.5.fasta)

Using 1 processors.

Reading in the pr2\_gb203\_version\_4.5.fasta template sequences… [ERROR]: template is not aligned, aborting.  
DONE.  
It took 0 to read 0 sequences.

Any help with this would be appreciated.

Regards,

Nicole

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**Author:** ![\_renh](https://avatars.discourse-cdn.com/v4/letter/_/ce7236/32.png) [@\_renh](https://forum.mothur.org/u/_renh)\
**Post date:** [August 7, 2017, 2:17pm UTC](https://forum.mothur.org/t/pr2-fasta-file-not-aligned/3357/2 "2017-08-07T14:17:46Z")

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Hi NicoleDames14,

the

```nohighlight
align.seqs

```

cmd ‘aligns a user-supplied fasta-formatted candidate sequence file to a user-supplied fasta-formatted template alignment’ (see the link: [https://www.mothur.org/wiki/Align.seqs](https://www.mothur.org/wiki/Align.seqs)). In your case the ‘user-supplied fasta-formatted candidate sequence file’ is ‘18S.unique.fasta’ and the ‘user-supplied fasta-formatted template alignment’ is ‘pr2\_gb203\_version\_4.5.fasta’

The problem is that the Protist Ribosomal Reference database (PR2) is not aligned, so you can not use it as ‘fasta-formatted template alignment’.

There are two options: (1) align the PR2 database first using a MSA (Multiple Sequence Alignment) tool, such as MAFFT or MUSCLE; or (2) skip the alignment step, i.e. it’s possible to go further with mothur pipeline using an alignment-independent approach.

I have been using the second option.

Basically, you skip that step and go directly to

```nohighlight
pre.cluster

```

. Then, with

```nohighlight
cluster

```

cmd you can use an alignment-independent clustering algorithm such as Vsearch dgc (method=dgc).

  
I think also, this question that you made was pointed out by another colleague here. Try to find it.  
I hope I have helped. Kind regards, @renh@

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<div class="post-metadata">

**Author:** ![NicoleDames14](https://avatars.discourse-cdn.com/v4/letter/n/ecccb3/32.png) [@NicoleDames14](https://forum.mothur.org/u/NicoleDames14)\
**Post date:** [August 8, 2017, 8:36am UTC](https://forum.mothur.org/t/pr2-fasta-file-not-aligned/3357/3 "2017-08-08T08:36:25Z")

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Thank you @renh@.

I realized now that pr2 is not aligned and should be used for taxonomy rather.

I have managed to figure out what to do, but will also I’ll try your suggested method.

Regards,

Nicole