Q&A in AGH635 Course: NTSys Application Part 1- Why Could Not I See All of My OTU’s in NTSys Output?

Incomplete OTU's in tree

Gbr. 1: Output hasil analisis NTSys untuk total 101 individu, tetapi mengapa hanya 30 saja yang ditampilkan dalam gambar output dari NTSys?

One of former S2 student participated in the 2010-AGH635 lecture ask me the following questions: “I did cluster analysis of my SSR data using NTSys. My samples consisted of 101 individuals (OTU’s), the SSR marker loci used were 14, and the total generated alleles were 130. After finishing my analysis and generating the output tree, why did the tree (Fig. 1) only display 30 OTU’s out of 101? How can I use NTSys to generate dendogram tree displaying all of 101 OTU’s instead of only 30?

Complete OTU

Fig. 2. The complete “Tree plot” containing 101 individuals (OTU’s) in one figure. However, the spacing among OTU’s were too closed and the OTU labels were not legible.

The answers to those questions were very simple, after you have generated dendogram tree that display only 30 OTU’s using NTSys – go to “OPTIONS” in the NTSys menu and select “PLOT OPTIONS…” New window – “TREE PLOT …” window will open. In the “TREE PLOT…” window, look for option  “Page Control” and “OTU’s/page” – it will by default contain number “30” (That is why you only see 30 OTU’s out of 101 displayed in the NTSys generated tree).

Still in that window, if you change the “OTU’s/page” number into “101” then your tree will display 101 OTU in one dendogram (Fig. 2). However, you will not be able to see the OTU’s labels legibly (in fact, you will not be able to discern what the labels are at all). In this case, you may want to add custom annotation using graphic editor. In Fig. 3, you should be able to see incomplete custom annotation (I purposely did not do it for all of your OTU’s) for the tree in Fig. 2. You may add your own annotation to suit the objective of your research. My interpretation of the tree the might be something like these:

Custo annotation

Fig. 3. The final “Tree plot” containing 101 individuals in one figure after adding custom annotations.

  • Sample #10 is mother plant and sample #10.1 – 10.9 are supposedly clonal propagation of sample #10. In such case, green block annotation indicated true clone of sample #10, while yellow block annotation may indicate some degrees of somaclonal variation.
  • Sample #61 is mother plant and sample #61.1 – 61.9 are supposedly clonal propagation of sample #61. In such case, green block annotation indicated true clone of sample #61, yellow block annotation may indicate some degrees of somaclonal variation, and red block annotation may indicate either heavily mutated seedlings or mislabeled/totally different seedlings labelled as clone of sample #61.
  • And so on, you are the one who know the interpretation of your data. That custom annotation may be an alternative of putting custon labels on NTSys tree that you can use to give more information.

Your next question might be “how can I have 101 OTU’s display in one tree, but still have meaningful label for the OTU’s?” Well… the answer will be to divide the tree for 101 OTU into 2-5 parts and combine them again using graphic editor. You will have good spacing among OTU’s and legible labels. However, your picture may take up more than one page long of a regular paper. Otherwise, you have to make a reduced copy of the printed picture (may be 2-3 pages in length) to make it fit into a standard (A4) paper.

Your further next question might be “how can I divide the generated tree for 101 OTU into 2-5 parts and combine them into one bigger picture using graphic editor?” The answer for this is go back to the stage where you have generated tree with 30 OTU using NTSys. Next – go to “OPTIONS” in the NTSys menu and select “PLOT OPTIONS…” New window – “TREE PLOT …” window will open. In the “TREE PLOT…” window, look for option “Page Control” and “OTU’s/page” – it will by default contain number “30.”

If you want to make 4 part of pictures, then change the number into “26” and you will have the first part of picture displaying the first 26 OTU’s in your computer monitor. Next – go to “FILE” in the NTSys menu and select “SAVE METAFILES…” New window – “SAVE PLOT AS METAFILES…” window will open. You have to give a name of your first part of your tree (i.e. “Tree Part-1”) and click “OK” to save your first part of tree picture.

Next – go to “OPTIONS” in the NTSys menu and select “PLOT OPTIONS…” New window – “TREE PLOT…” window will open. In the “TREE PLOT…” window, look for option “Page Control” and “Page” – it will by default contain number “1.” If you change the number into “2” then you will see the second part of your tree. Similarly, if you change it into “3” or “4” then it will display the third and the fourth part of your tree. Each time the second, the third, and the fourth parts of the picture are displayed, save the picture using “FILE” and “SAVE METAFILES…” steps in the above section.

Once you have gotten all parts of the picture, then import the “.wmf” files of the picture parts into graphic editor and joint them into one complete picture. If you ask me “how can I edit the picture using graphic editor?” Well… that question is a Rp. 250 thousand an hour tutorial workshop question, so you better not ask me about it.

I hope that those explanations are able to answer your questions and solve your problems. Otherwise, feel free to contact me and ask for more questions to clarify parts that you may not get them. Best of luck in your study and data analysis. I hope you will be able to complete your S2/S3 program at IPB soon.

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About PMB Lab: Prof. Sudarsono

This blog is dedicated as a communication media among alumni associated with PMB Lab, Dept. of Agronomy and Horticulture, Fac. of Agriculture, IPB, Bogor – Indonesia. It contains various information related to alumni activities, PMB Lab’s on going activities and other related matters.
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