[ERROR]: template is not aligned, aborting in mothur454 analysis

Commands in mothur
1

Dear Pschloss,
Thanks for help in understanding 454SOP. I have started to work on my sample(which contains fungal sample) data from 454seq. When i am doing alignment using command

mothur > align.seqs(fasta=its.shhh.trim.unique.fasta, reference=UNITEv7_sh_99.fasta, inputdir=ITS, outputdir=ITS, processors=12)
Setting output directory to: ITS/
Setting input directory to: ITS/

Using 12 processors.

Reading in the ITS/UNITEv7_sh_99.fasta template sequences… [ERROR]: template is not aligned, aborting.
DONE.
Where is the problem, Iam not able to sort it out…

Thanks in advance.

2

I’m pretty sure that the UNITEv7_sh_99.fasta file is not aligned. Frankly, I don’t think you can meaningfully align ITS fragments since they are not homologous across taxa (or even within a taxon).

Pat

3

Thanks Pschloss… then which reference file we can use to align fungal sequence…