Each and All of Sequences adressed to indiviuals OTU, how can I solve this?

Theory behind mothur
1

I have close to 130000 Sequences and when I use mothur to classify and obtain taxonomic information. I got full taxonomy for them all which seems accurate. But Each and all of Sequences adresses to indiviuals OTUs (I have close to 130000 OTUs), how can I solve this? where did I do wrong and here is my commands;

unique.seqs(fasta=.fasta)
align.seqs(candidate=.unique.fasta,template=a template file,flip=t,processors=12) filter.seqs(fasta=.unique.align) unique.seqs(fasta=.unique.filter.fasta,name=.names) dist.seqs(fasta=.unique.filter.unique.fasta,calc=onegap,countends=F,cutoff=0.03,output=lt,processors=12) cluster(phylip=.unique.filter.unique.phylip.dist,name=.names,method=average,cutoff=0.03)
classify.seqs(fasta=.fasta,template=a non gap template file,taxonomy=**.tax,cutoff=60) classify.otu(taxonomy=.taxonomy,list=*.unique.filter.unique.phylip.fn.list,label=0.03)

2

What are you sequencing? Can you do summary.seqs on your aligned fasta and copy the results here?

3

Start End NBases Ambigs Polymer NumSeqs
Minimum: -1 -1 0 0 1 1
2.5%-tile: 1855 2578 272 0 4 6139
25%-tile: 1903 2583 272 0 4 61383
Median: 1903 2632 274 0 5 122766
75%-tile: 1903 3726 274 0 5 184149
97.5%-tile: 1904 3732 274 0 6 239393
Maximum: 6852 6852 274 0 57 245531
Mean: 1908.5 3122.04 272.162 0 4.68964

of Seqs: 245531

4

16s rrna by the way

5

I need your help guys

6

But Each and all of Sequences adresses to indiviuals OTUs (I have close to 130000 OTUs), how can I solve this?

Sorry, but I don’t understand what you’re asking.

7

To start with, with your line

dist.seqs(fasta=*.unique.filter.unique.fasta,calc=onegap,countends=F,cutoff=0.03,output=lt,processors=12)

Can you try running it again with a higher cutoff? It’s explained in the FAQs here, but basically the cutoff during distance calculation isn’t the same as the distance you later cluster at. Usually the maximum distance in your matrix will be lower than the cutoff you use during the distance matrix.

8

I have ~130000 sequences and I have ~130000 OTUs. Each of my sequences annotated as individual OTUs. I think now it is more clear :slight_smile:

9

At what OTU label? Have you tried the higher cutoff in dist.seqs as suggested by dwaite?