Align.seq was successful with the older mothur version but not with v1.48.0

1

I reran the MiSeq SOP today with the same sequences and reference as I did in March/April this year. It was successful back then, but this time I encountered problems with align.seq.

The error: [ERROR]: template is not aligned, aborting.

My sequences are 23S rDNA and I used this database (http://microgreen-23sdatabase.ea.inra.fr/downloads/microgreen-db_v1.1/1_AlgaeBase/microgreen_algaebase_mothur.fasta) as reference.

I noticed that the new release (1.48.0) was in May, which was after my successful trial in March/April. Is the reference database not compatible with the new mothur version?

2

Hi

It isn’t mothur - it’s your reference sequences. When I go to the link you provided those sequences are not aligned. They have to be aligned, as the error message says, to be used with align.seqs. I suspect you were using a different reference last May. It doesn’t look like the microgreen database provides an aligned version.

Pat

4

Hi Pat,
Thanks for the information.
Do you have an aligned 23S rRNA database to share? My sequences are from the domain V of the 23s rRNA gene. I tried to identify algal species from environmental samples. I tried to extract eukaryotes and cyanobacteria using get.lineage from Silva LSURef 138.2 as a reference.
But I got the below warning:

get.lineage_25%_ambiguous
get.lineage_25%_ambiguous1895×383 39.6 KB

before get.lineage, the sequence looks like this

nt U
nt U972×579 5.65 KB

after get.lineage, the sequence looks like this

nt N
nt N1890×548 11.2 KB

I’m guessing it’s because U instead of T was used in the original reference and U was replaced with N after get.lineage? How could I quickly replace U with T, if it is the right correction?

Best,
Yan

5

Hi,

Sorry, I don’t have anything for 23S. You could do it in in R or with a sed command from the linux command line.

Pat

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