# custom reference alignment for functional genes **URL:** \ **Category:** Theory behind mothur\ **Created:** [January 14, 2013, 8:59am UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176 "2013-01-14T08:59:30Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![Kirk](https://avatars.discourse-cdn.com/v4/letter/k/f9ae1b/32.png) [@Kirk](https://forum.mothur.org/u/Kirk)\ **Post date:** [January 14, 2013, 8:59am UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/1 "2013-01-14T08:59:30Z")
Dear, I was wondering if the reference alignment should have a specific format? I’ve alway been using 16S sequences, but now I started with funcional genes. I’ve compiled a reference alignment using MEGA, but when I try to use it, I get an error message [ERROR]: template is not aligned, aborting. Does it need some additional formatting, although I can’t immediately see what could be wrong? e.g. ```nohighlight >YP_001167793.1_Rhodobacter_sphaeroides_ATCC17025 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATGTTTACCCGCCGCGCCGCC------------------------------------------------------------------------------------------------------------------------------------------------------------------CTCGTGGGAGCCGCCGCGCTCGCGTCAGCG---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CCGCTCGTCATCCGGACGGCAGGCGCCGAGGAAGCTCCGGCCCAGCTTGCCAGTGCAGCCCCCGTCGATCTCAGCAACCTGCCCCGC---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GTGAAGCACACG------CTCGTGCCTCCGCCCTTCGCGCATGCCCACGAGCAGGTGGCCGCCAGC------------------------------------------------------------------------------GGC---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CCCGTCATC------AAC---GAATTCGAGATGCGGATCATCGAGAAGGAA---GTGCAGCTCGAC---GAA---GATGCCTACCTGCAGGCGATGACCTTCGAC------------------------GGCTCGATCCCC---------GGCCCGCTGATGATCGTGCATGAGGGCGACTATGTCGAACTCACCCTGATCAACCCGCCCGAGAACACC---------------------------------------------------------------------------------------------ATGCCCCACAACATCGACTTCCACGCCGCCACCGGCGCGCTG------------GGAGGCGGCGGGCTCACGCTC---------ATCAATCCGGGCGAAAAGGTCGTCCTGCGGTTCAAGGCCACGCGCGCGGGCGCCTTCGTCTATCACTGCGCCCCCGGCGGCCCGATG---ATCCCCTGGCACGTCGTC---GGCGAGTGGGACAACGACCTGATGGAACAGGTCGTGGCGCCGGTCGGCCTCACCGGCTGA----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ``` thanks ---
**Author:** ![westcott](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/westcott/32/18_2.png) [@westcott](https://forum.mothur.org/u/westcott)\ **Post date:** [January 14, 2013, 2:53pm UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/2 "2013-01-14T14:53:44Z")
Mothur gives that warning when the sequences are not all the same length. Can you run summary.seqs on your template and post the results? ---
**Author:** ![Kirk](https://avatars.discourse-cdn.com/v4/letter/k/f9ae1b/32.png) [@Kirk](https://forum.mothur.org/u/Kirk)\ **Post date:** [January 14, 2013, 3:12pm UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/3 "2013-01-14T15:12:23Z")
Hi, I’ve realigned the references and now it works. Thanks ---
**Author:** ![Ghazal](https://avatars.discourse-cdn.com/v4/letter/g/958977/32.png) [@Ghazal](https://forum.mothur.org/u/Ghazal)\ **Post date:** [February 3, 2015, 10:48am UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/4 "2015-02-03T10:48:25Z")
Hello with special thanks to you for your great software, I have exactly the same problem. I am using “gold” as my reference and whatever I did, the mothur gives me the error: [ERROR]: template is not aligned, aborting. I have used this reference before a lot and this is the first time I encounter this error. This is the summary of my template: mothur \> summary.seqs(fasta=gold.fasta) Using 1 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 1205 1205 0 4 1 2.5%-tile: 1 1335 1335 0 5 260 25%-tile: 1 1445 1445 0 5 2591 Median: 1 1476 1476 0 5 5182 75%-tile: 1 1507 1507 2 6 7772 97.5%-tile: 1 1550 1550 20 7 10103 Maximum: 1 1655 1655 30 16 10362 Mean: 1 1469.86 1469.86 2.26809 5.55761 # of Seqs: 10362 would you please help me? Thanks Ghazal ---
**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:** [February 3, 2015, 1:40pm UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/5 "2015-02-03T13:40:29Z")
Where are you getting the gold.fasta file from? The silva-formatted version is available here: [http://www.mothur.org/w/images/f/f1/Silva.gold.bacteria.zip](http://www.mothur.org/w/images/f/f1/Silva.gold.bacteria.zip) ---
**Author:** ![Ghazal](https://avatars.discourse-cdn.com/v4/letter/g/958977/32.png) [@Ghazal](https://forum.mothur.org/u/Ghazal)\ **Post date:** [February 6, 2015, 10:23am UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/6 "2015-02-06T10:23:20Z")
Actually I am replicating another study and have to use gold.fasta as they have used it before. Anyway, thanks a lot for your reply. ---
**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:** [February 6, 2015, 12:53pm UTC](https://forum.mothur.org/t/custom-reference-alignment-for-functional-genes/1176/7 "2015-02-06T12:53:24Z")
I suspect our version of the gold is the same as theirs. Pat