Dear Pat,
I am a beginner in MOTHUR, & i hope you will guide me.
I have 300 clones subjected to RFLP analysis. Only one unique clone is sequenced for each RFLP pattern. THerefore, i only have a sequence for a handful of clones, but i know the number of times that this RFLP pattern appears.
I have checked out http://www.mothur.org/wiki/Read.dist#name
I would like to assign sequenced clones to their respective OTUs based on >97% similarity.
What I have done:
Subjecting unique sequences to be aligned in MOTHUR.
Created distance matrix
What i want to do:
Include “names file”
How do I create a names file for 16S amplified ribosomal DNA Restriction analysis, as i would also like to use it for downstream processing:
- Rarefaction curve
- calculation of diversity indices
Thank you.
Hi there,
Creating a names file is fairly straight forward. The basic format is the first column contains the sequence name and the second column contains the names of the redundant sequences separated by a comma. So you could either use your clone names or make something up. For example…
Sequence1 Sequence1, A, B, C
Sequence2 Sequence2, D
Sequence3 Sequence3, E, F, G, H
Sequence4 Sequence4
etc…
Save all of this in a text file and you should be good to go.
Pat
Hi there!
I have uploaded my fasta file with e.g seq1, seq2, seq3, seq3, seq4 and seq5 (total = 5 sequences)
Considering i have a handful of clones for a particular sequence, i’ve prepared a name file:
seq1 seq1,seq1.1,seq1.2,seq1.3
seq2 seq2,seq2.1,seq2.2
seq3 seq3,seq3.1,seq3.2,seq3.3,seq3.4
seq4 seq4
seq5 seq5,seq5.1
(total number=15 clones)
Now, I require a group file.
what i’ve done: >make.group(fasta=x.fasta, groups=A)
This created a Group file which looked like:
Seq1 A
Seq2 A
Seq3 A
Seq4 A
Seq5 A
I proceeded with >make.shared(list=x.list, group=A.groups)
This then appeared:
seq1 is in your listfile and not in your groupfile. Please correct
seq1.1 is in your listfile and not in your groupfile. Please correct
…seq5.1 is in your listfile and not in your groupfile. Please correct
I have also tried to create a Group file on excel (text file) which looked like:
Seq1 A
Seq1.1 A
Seq1.2 A
Seq1.3 A
…
Seq5.1 A
and proceeded with >make.shared(list=x.list, group=A.groups)
Now, this appeared:
seq1 is in your group file and not in your list file. Please correct
seq1.1 is in your group file and not in your list file. Please correct
…seq5.1 is in your group file and not in your list file. Please correct
How do i create a compatible group file??
Thank you!
I have done:
dist.seqs(fasta=x.fas, output=lt)
cluster(phylip=…, cutoff=0.03, name=x.names)
Why is it that
>summary.single(label=0.01-0.03-0.05)
showed:
Your file does not include the label 0.01. I will use unique
Your file does not include the label 0.03. I will use unique
Your file does not include the label 0.05. I will use unique
I also wish to classify OTU at >97% similiarity.
classify.otu(taxonomy=…, name=…, list=…, label=0.01-0.03-0.05)
Again,
Your file does not include the label 0.01. I will use unique
Your file does not include the label 0.03. I will use unique
Your file does not include the label 0.05. I will use unique
Please point out where went wrong.
Many thanks!
seq1 is in your group file and not in your list file. Please correct
seq1.1 is in your group file and not in your list file. Please correct
…seq5.1 is in your group file and not in your list file. Please correct
These sequence names are in lowercase font whereas above you had the “S” in upper case. Are you being consistent with the shift key between files? mothur is case-sensitive this way.
Dear pat, Yes,the shifts/case in between files were the same. But did not work! Is there something wrong with my input? Also, why am I not able to get otu at 0.01,0.03 etc. It generated only UNique otu which defeated the purpose of otu classification?! Thanks!
Can you email your files to mothur.bugs@gmail.com?
Is it possible that your sequences are actually quite different from each other and wouldn’t cluster at those levels?
Pat