align.seqs command killed after sometime

Hi Everyone,
I have a 16s DNA metagenomic data and I am using align.seqs command to align the reads to ribosomal database. I have downloaded aligned sequences from the ribosomal database. When I am running the command after sometime it get killed, I think because it is using lot of memory. I do not know how to solve this issue, please anyone can help me in this.
Thanks for any help!!!

Can you post the command text you are using? How much RAM does your computer have?

Thanks for looking into my problem, the command I am using is
align.seqs(fasta=…/demultiplexed.fna, reference=…/rdp.fa, flip=T,search=blast, align=blast)
I am doing this on server which has 32Gb of memory.
The rdp database is around 68 Gb, this is the aligned database I am using, before I used unaligned database and it was fine but when I was using filter.seqs command after aligning the reads. It says that the sequence are of unequal length.
Can you suggest any other database for 16s DNA alignment.

So you’re saying that rdp.fa is not aligned? That’s not going to work…

When I was using unaligned database then it was going fine, but now I am using aligned database and it is getting killed every time.
The database is very big itself.


rdp.fa is aligned

If I had to guess, 68GB won’t fit into 32GB of RAM. Why not use our silva.bacteria.fasta reference? It’s a lot better than the RDP…

Thanks for the reply Schloss,
I used greengenes database for alignment, but when I am using filter.seq command then it is giving me error that sequence are not of the same length.
is that database not aligned.
I downloaded it from here

Not sure what you were using… Use this:

Thanks Pat,
I am now using this.

Hi Pat,
I used Silva database as suggested by you, my reads got aligned and then I did the next step which is filtering the sequences by using command filter.seqs(fasta=…/demultiplexed.u.align, vertical=T, trump=., processors=2) When I am running this command I am getting this error can you tell me why I am getting this error. I thought when the databases are not aligned we get this type of errors.
The error is:
Creating Filter…
Sequences are not all the same length, please correct.
Sequences are not all the same length, please correct.
My summary file looks like this:
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: -1 -1 0 0 1 194
25%-tile: 13862 21777 151 0 4 1933
Median: 13862 21777 151 0 4 3865
75%-tile: 13862 21777 151 0 5 5797
97.5%-tile: 13870 21780 151 0 6 7535
Maximum: 15883 22438 151 0 8 7728
Mean: 13451.4 21117.5 146.075 0 4.28429

of Seqs: 7728

Please help!!!

can you provide the exact commands and ordering you are using (and version of mothur you are using)?

Hi Pat,
The command I used is
filter.seqs(fasta=…/demultiplexed.u.align, vertical=T, trump=., processors=2)

I did not screened the sequences using screen command. But, once I did screen.seqs I do not get the error:
The screen command is
screen.seqs(fasta=…/demultiplexed.u.align, start=13862, end=21777, maxhomop=6)

I hope I am going on the right path.
Also, when I aligned my sequences with archea I get this summary file :
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: -1 -1 0 0 1 194
25%-tile: -1 -1 0 0 1 1933
Median: -1 -1 0 0 1 3865
75%-tile: -1 -1 0 0 1 5797
97.5%-tile: -1 -1 0 0 1 7535
Maximum: 14974 21780 151 0 5 7728
Mean: 81.8675 128.375 0.876423 0 1.0198

of Seqs: 7728

Does this mean that none of my sequences got hit in archae.
The mothur version I am using is mothur v.1.27.0