# Difficulty customizing my reference alignment using align.seqs

**URL:** <https://forum.mothur.org/t/difficulty-customizing-my-reference-alignment-using-align-seqs/3376>\
**Category:** Commands in mothur\
**Created:** [August 24, 2017, 3:03am UTC](https://forum.mothur.org/t/difficulty-customizing-my-reference-alignment-using-align-seqs/3376 "2017-08-24T03:03:11Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![vicentejan](https://avatars.discourse-cdn.com/v4/letter/v/a6a055/32.png) [@vicentejan](https://forum.mothur.org/u/vicentejan)\
**Post date:** [August 24, 2017, 3:03am UTC](https://forum.mothur.org/t/difficulty-customizing-my-reference-alignment-using-align-seqs/3376/1 "2017-08-24T03:03:11Z")

</div>

I want to determine the start and end positions of primers that I used to target the D1 region of the 28S rRNA. I downloaded the LSU Parc file from the SILVA data base and generated a silva.full\_v128.fasta following the “README for the SILVA v128 reference files” instructions.

I generated a Munguiculata.pcr.fasta file with the region targeted for both of my primers.

When I run the command:

align.seqs(fasta=Munguiculata.pcr.fasta, reference=silva.full\_v128.fasta, processors=8)

I get the message

“Unable to open Munguiculata.pcr.fasta. Trying output directory /home/vicente/Desktop/mothurbioerosion/Munguiculata.pcr.fasta  
Using 8 processors.  
Reading in the silva.full\_v128.fasta template sequences… Killed”

At this point, I am not sure whether there is a problem with either file or if the sequence Munguiculata.pcr.fasta is not present in the “silva.full\_v128.fasta” file.

Any help would be greatly appreciated.

Thank you,  
Jan

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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:** [August 25, 2017, 5:43pm UTC](https://forum.mothur.org/t/difficulty-customizing-my-reference-alignment-using-align-seqs/3376/2 "2017-08-25T17:43:57Z")

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Within mothur, if you run…

For windows…

```nohighlight
system(dir)

```

For Mac/Linux

```nohighlight
system(ls)

```

Do you see Munguiculata.pcr.fasta or silva.full\_v128.fasta? If so, what type of computer are you running this on and how much RAM does it have?

Pat

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<div class="post-metadata">

**Author:** ![vicentejan](https://avatars.discourse-cdn.com/v4/letter/v/a6a055/32.png) [@vicentejan](https://forum.mothur.org/u/vicentejan)\
**Post date:** [September 12, 2017, 9:52pm UTC](https://forum.mothur.org/t/difficulty-customizing-my-reference-alignment-using-align-seqs/3376/3 "2017-09-12T21:52:43Z")

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Hi Patt,

Im running my commands on Ubuntu 15.10 but realized I didn’t have enough RAM. I created a SWAP file and now everything seems to work. ummary.seqs(fasta=Munguiculata.pcr.align, processors=12)

Using 12 processors.

Start End NBases Ambigs Polymer NumSeqs  
Minimum: 69157 72701 357 0 5 1  
2.5%-tile: 0 0 0 0 0 1  
25%-tile: 0 0 0 0 0 1  
Median: 0 0 0 0 0 1  
75%-tile: 0 0 0 0 0 1  
97.5%-tile: 0 0 0 0 0 1  
Maximum: 69157 72701 357 0 5 1  
Mean: 69157 72701 357 0 5

# of Seqs: 1

Output File Names:  
/media/drive\_2/Jan/mothurbioerosion/Munguiculata.pcr.summary

Thank you!  
Jan