# Customize coordinates for v4 region **URL:** \ **Category:** Commands in mothur\ **Created:** [May 16, 2023, 2:52am UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825 "2023-05-16T02:52:41Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![zhinew](https://avatars.discourse-cdn.com/v4/letter/z/71e660/32.png) [@zhinew](https://forum.mothur.org/u/zhinew)\ **Post date:** [May 16, 2023, 2:52am UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/1 "2023-05-16T02:52:41Z")
Hello, I am customizing reference alignment for 16S v4 region. After I align the .fasta file to the silva.seed\_v132.align file using “align.seqs” command, there was no .align file being generated and I can’t run the summary.seqs command. Can anyone help me with this issue? ---
**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:** [May 16, 2023, 6:00pm UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/2 "2023-05-16T18:00:24Z")
Can you post the commands you are running and the names of the files that are being generated along with any warning/error messages? Thanks, Pat ---
**Author:** ![zhinew](https://avatars.discourse-cdn.com/v4/letter/z/71e660/32.png) [@zhinew](https://forum.mothur.org/u/zhinew)\ **Post date:** [May 16, 2023, 7:15pm UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/3 "2023-05-16T19:15:23Z")
Hi Dr. Schloss, This is the my align command: “align.seqs(fasta=Ecoli.v4.fasta, reference=silva.seed\_v132.align)” There is no .align file generated after this command, maybe something wrong with my v4.fasta file and I can post later. Alternatively, I just use v4 coordinates 11895 and 25318 to trim the silva.seed\_v132.align and it worked fine. Two quick questions: Should I use the Silva full-length sequence references or the recreated seed database as the alignment reference? Can I use the HMP\_MOCK.v35.fasta file to check my own sequencing errors? Thanks for your time. ---
**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:** [May 18, 2023, 5:30pm UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/4 "2023-05-18T17:30:32Z")
Hi, 1. Were there any error messages when you ran `align.seqs`? 2. I think the recreated seed should work as well as the non-redundant alignments (they both contain full length sequences) 3. I doubt you sequenced the HMP Mock community. You would need to get your own fasta file with sequences for the 16S rRNA gene sequences in your mock comunity Pat ---
**Author:** ![zhinew](https://avatars.discourse-cdn.com/v4/letter/z/71e660/32.png) [@zhinew](https://forum.mothur.org/u/zhinew)\ **Post date:** [May 18, 2023, 6:21pm UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/5 "2023-05-18T18:21:19Z")
Hi Dr. Schloss, 1. When I ran align.seqs, nothing popped out . 2. Yes, the recreated seed file worked fine for me now. 3. I found my own mock file. However, when I used them as reference to run the error.seqs command, the following warning message popped out. Maybe I should screen the mock file to get rid of ambigs? [WARNING]: We found more than 25% of the bases in sequence AA\>1BCAFFAC11FEGFG?FGHCG?AEHHHHF1FCEGEGHGGEFEEEEGECGEC\>ECDGGHHFFFGBFHHEG?GCEFGH?EGE\<\>?GFFGCGG/\>@CGCFFHH1C1FGB11GFCGECCG0\<0=0DGGFHCCGEHGHGHHGEHHHHHGHFEGFBFGG?.;EFB0.9AB-99////;9FFA–B?FFFF9BB/-@\>-999=@@?FFFFFFBBFFFF//;///—?BF99AA–;-BFB–@-@=9A@9\>@- to be ambiguous. Mothur is not setup to process protein sequences. [WARNING]: 417 reference sequences have ambiguous bases, these bases will be ignored. ---
**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:** [May 23, 2023, 5:44pm UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/6 "2023-05-23T17:44:17Z")
I suspect you haven’t correctly formatted your reference file. ---
**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:** [June 2, 2023, 5:45pm UTC](https://forum.mothur.org/t/customize-coordinates-for-v4-region/21825/7 "2023-06-02T17:45:02Z")
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