# Customize Silva reference for V4 region **URL:** \ **Category:** Commands in mothur\ **Created:** [July 28, 2023, 2:03am UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892 "2023-07-28T02:03:34Z")\ **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:** [July 28, 2023, 2:03am UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/1 "2023-07-28T02:03:34Z")
Hi guys, I used primer 515F and 806R to select the v4 region from the E coli 16S rRNA sequence list on the mothur SOP, and align the v4 region to silva.seed\_v132.align. However, the summary showed that the start and end position are both -1. Does anyone know what could be wrong here? ---
**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:** [July 31, 2023, 5:56pm UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/2 "2023-07-31T17:56:24Z")
Hmmm, I’m not sure what you’re doing to generate the custom alignment. Can you provide more detail? For what it’s worth, the numbers in the MiSeq SOP are for the V4 region. You should be able to use those without a problem. Pat ---
**Author:** ![zhinew](https://avatars.discourse-cdn.com/v4/letter/z/71e660/32.png) [@zhinew](https://forum.mothur.org/u/zhinew)\ **Post date:** [August 2, 2023, 3:03pm UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/3 "2023-08-02T15:03:44Z")
Hi Dr. Schloss, I was just trying to see if I can customize the Silva seed reference by myself, because I might need to use reference alignment for other regions. I used primer 515F GTGYCAGCMGCCGCGGTAA and 806R GGACTACNVGGGTWTCTAAT to trim the E coli 16S rRNA sequence provided on the Miseq SOP, and I aligned this V4 sequence against silva.seed\_v132.align, and here is the summary report. Thanks for your kind help! ![2023-08-02](https://canada1.discourse-cdn.com/flex036/uploads/mothur/original/1X/c7c33c6b1ccf9559a4ac9ebba3d4ccf215bcc64d.png) ---
**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 3, 2023, 7:11pm UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/4 "2023-08-03T19:11:40Z")
It would be helpful to see all of the commands you are running to get to this point. Pat ---
**Author:** ![zhinew](https://avatars.discourse-cdn.com/v4/letter/z/71e660/32.png) [@zhinew](https://forum.mothur.org/u/zhinew)\ **Post date:** [August 3, 2023, 7:33pm UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/5 "2023-08-03T19:33:25Z")
Hi Dr. Schloss, please see the following commands: mothur \> align.seqs(fasta=Ecoli.v4.fasta, reference=silva.seed\_v132.align) Using 40 processors. Reading in the /home/zhinew//UMich whole genome sequencing project/SILVA database/silva.seed\_v132.align template sequences… DONE. It took 7 to read 11180 sequences. Aligning sequences from /home/zhinew//UMich whole genome sequencing project/SILVA database/Ecoli.v4.fasta … 1 It took 0 secs to align 1 sequences. It took 0 seconds to align 1 sequences. Output File Names: /home/zhinew//UMich whole genome sequencing project/SILVA database/Ecoli.v4.align /home/zhinew//UMich whole genome sequencing project/SILVA database/Ecoli.v4.align\_report mothur \> summary.seqs(fasta=Ecoli.v4.align) Using 40 processors. ``` Start End NBases Ambigs Polymer NumSeqs ``` Minimum: 0 0 0 0 1 1 2.5%-tile: 0 0 0 0 1 1 25%-tile: 0 0 0 0 1 1 Median: 0 0 0 0 1 1 75%-tile: 0 0 0 0 1 1 97.5%-tile: 0 0 0 0 1 1 Maximum: -1 -1 0 0 1 1 Mean: 0 0 0 0 1 # of Seqs: 1 It took 0 secs to summarize 1 sequences. ---
**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 3, 2023, 7:53pm UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/6 "2023-08-03T19:53:43Z")
Can you post the contents of these two files? Ecoli.v4.fasta and Ecoli.v4.align Pat ---
**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:** [August 13, 2023, 7:54pm UTC](https://forum.mothur.org/t/customize-silva-reference-for-v4-region/21892/7 "2023-08-13T19:54:26Z")
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