Definition of singleton, doubleton and quadrupletons in OTU


I want to ask a basic question regarding OTU.

What is the meaning of singleton, doubleton, tripleton and quadrupletons in OTU?

I am very new in this field, I had 351 16S rRNA clone library sequences for oil palm plantation soil sites. I want to make rarefaction curve from my samples. So now I still learning about how to do it using Mothur.

Can anyone help me with these definitions?

I really appreciate it.

Thank you so much

  • Nor Hashimah

Singleton - an OTU with only one sequence. Doubleton - an OTU with two sequences, etc…

Thank you so much.

May I know how can if OTU is singleton, doubleton, etc?

You can look in the list file or use the sabund file - the first column is the cutoff, the second column is the number of sequences in the largest OTU. Then comes the number of singletons, doubletons, etc.

Thank you so much for your help.

I have one more question.

For align.seqs command, after I used it for my sample, it showed this :

Some of you sequences generates alignments that eliminated too many bases, a list is provided in Sample1.unique.flip.accnos.
If you set the flip parameter to true mothur will try to aligning the reverse compliment as well.

From above comment, does it mean, I need to reverse compliment my sequences first before try to use align.seqs command?

Thank you so much

Some sequences align poorly and give that notice because they end up getting trimmed. If you know your sequences are in the correct (5’ to 3’) direction, then don’t worry about it, they’ll get junked in the screen.seqs command. If most/all of your sequences end up in the flip.accnos file then you should probably have flipped them back in trim.seqs. You can always just run align.seqs wiht flip=T as the message says.


Thank you so much, Prof.Pat

The more I learned, the more I’m interested in mothur!

I would like to ask another question.

I want to have Shannon index data and Evenness index data for my samples. Before this, I just try using command :
rarefaction.single(list=Sample1RU.fn.list, calc=shannon)

Please correct me if I’m wrong.

But when I tried to view the data, I’m confused.

So I tried to read more in Shannon calculator (Mothur’s wiki), and I tried to use this command :
mothur > summary.single(sabund=98_lt_phylip_amazon.fn.sabund, calc=shannon)

And I got the data, I would like to use 0.03 cutoff data to put inside my table.

Which data should I put, the shannon_lci or shannon_hci?
What is the differences and the reason?

Thank you so much

You want just shannnon - the _lci and _hci are the bounds on the 95% confidence interval

You mean, this data? (The one that I bold with yellow color)

Thank you so much!

Can I know how can I calculate Shannon evenness and Simpson evenness. I tried to look up at
but there is nothing info in the pages.

Thank you so much again!

Those are the ones. I’d suggest looking at Anne Magurran’s book on quantitative ecology

Thank you so much.

One more question, to use make.shared command, I need to have multiple set of sample in one file. Can I know how to put 3 sample sequences into one file before use make.shared command?

Thank you again

RIght - Ideally, you need to be processing all of the sequences from your 3 samples together from the beginning. Please see the SOP page for details on how this is done…

Thank you so much.

I managed to gain the data.

I have one question regarding Libshuff.

After I got the coverage and summary result,

This is the result for coverage :

I want to plot the graph of libshuff like in your publication.

I tried to figure out how to plot it.

From the Libshuff website : it stated that :

Q: How do you create the graphs using the data in the “coverages” and “results” output files?
A: We have used a number of graphing programs, including CricketGraph, DeltaGraph, and Microsoft Excel. To create graphs similar to those in our publications, place “d” on the X-axis, “X” and “XY” on the Y-axis, and “diff-X/XY” and “95% XY” on the secondary Y-axis. To plot the reciprocol comparison, simply substitute the data in “Y”, “YX”, “diff-Y/YX”, and “95% YX” on the proper axes

May I know if I which one considered as X? Is that my sample A, B and C?

And which one is XY, diff-X/XY and 95%?

Thank you so much for your kind help!

In the future please split your questions into separate posts so each post has a theme - we’re pretty far from your original question…

If you have A, B, and C, then for the first comparison you would have A vs. B (X vs. Y) and B vs A (Y vs X). You’d repeat this for each pair (AB, AC, and BC).


I’m sorry coz I’m afraid I have a lot of question. After this I will make new post each time I ask different related question.

Again, thank you so much for explaining~