custom reference alignment for functional genes

Theory behind mothur
1

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.

>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

2

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?

3

Hi,

I’ve realigned the references and now it works.

Thanks

4

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

5

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

6

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.

7

I suspect our version of the gold is the same as theirs.

Pat