In order to be simple and clear, the relative percentage brightness data of DGGE bars obtained after the matching by Quantityone or similar software are sorted into excel tables. The format is as follows, that is, the sample numbers are arranged vertically from top to bottom, and the band numbers are arranged from left to right in the first line. Fill in the table with the relative percentage brightness values of the corresponding bands. For example, the brightness of band 3 in the 1 channel is 0.59 184 1. This table is part of the original data of one of my papers.

Step 2, import DGGE map data.

Canoco software was used for correlation analysis. Canoco has downloaded the cracked version in many forums, such as planta. When everyone is running, just adjust the computer system time to 2002 (the latest cracked version doesn't seem to need so much trouble). Canoco cannot directly identify data in excel format. The strip data of DGGE map must be converted into recognizable files. Dta suffix, and then the subsequent PCA and other related analysis can be carried out. The specific operation is as follows: in the excel table, use the mouse to select a piece of data together with the sample number and the piece number, and use CTRL+C to copy it. Then, under Start-Program, find the subroutine WcanoImp of Canoco, which is specially used for importing data. The program will pop up the following dialog box:

If your bar matrix data has many zero values, you can check the last "Save in compressed format". As for why, I won't say much. But if you don't check it, it doesn't seem to affect the analysis results.

Click Save to open the following dialog box:

At this time, you should choose the storage path of the strip data matrix and name the file. For example, I saved this file in the "PCA Tutorial" folder and named it spe.dta. Note that this "spe.dta" was typed by myself. Click Save to open the following dialog box:

Click OK, and you will be prompted to generate the file successfully. This file is the band composition data that Canoco software can identify, and it is used for our subsequent PCA analysis. This spe.dta file is saved in the PCA Tutorial folder. Of course, we can also do the same for the data of physical and chemical factors. If you are interested in the study of the impact of species' living environment on organisms, you can carry out analysis such as RDA and CCA, which requires two data matrices: species and environment.

The third step is to analyze PCA data with Canoco software.

Next, we can use Canoco software to analyze the memory PCA of our spe.dta file.

Find Canoco for Windows 4.5 under Start-Program (of course, your version may be different from mine).

Turn off the small advertisements that pop up in Canoco software. Enter the analysis interface of Canoco:

Click on the leftmost symbol ""to pop up the following dialog box:

We only have banded (that is, species category) data, so we can only choose "only category data available" at the top. If you have species data, physical and chemical parameter data of the environment in which the species lives or other auxiliary data, you can choose the corresponding option. Different options, the subsequent operation is slightly different, and different analysis can be made. For example, "available species and environmental data", we can analyze RDA and CCA to calculate the influence of different environmental factors on species composition. Interested parties can consult the literature.

Select "Next":

Browse, browse and select the "spe.dta" data generated in the previous step. As follows:

Then, in the column of "Canoco solution file name", copy the storage path of "spe.dta" data. For example, this time I will copy the blue selection in the figure below, then copy it to "Canoco solution file name", and then enter the file name of the solution to be generated in the future, such as PCA.sol, paying attention to the suffix. Saul.

This step is more important.

Select "Next":

Select PCA in linear analysis mode, and then select Next:

By default, next:

If your data in different bands are very different, for example, the largest is 100, and the smallest is 0. 1, you can choose the square root. By default, next:

By default, next:

By default, next:

Select "Finish" and confirm that Canoco software analysis is completed. The following dialog box pops up:

In the File Name column, type PCA.con As shown below. Of course, you can choose any name, and the suffix is best. Disadvantages.

The following depends on the analysis results and drawings.

The fourth step is to analyze the results and draw the Canoco PCA data.

After selecting Save in the previous step, the following dialog box will pop up:

Select Analyze to analyze the data.

We can see the approximate analysis results of PCA, as shown in the figure below. We can see that the first axis explains the species composition change of 3 1.3%, and the second axis explains the species composition of 46.9-3 1.3 = 15.6%.

It's time to draw! Select the "CanoDraw" button to pop up the following dialog box named PCA.cdw.

Then "save". Then you can output the picture. First, turn off the Canoco advertisement below.

We can see such an operation interface:

Select "scatter plot"-"sample" or "species" under the "Create" menu to get the PCA diagram of sample points or species composition. As shown in the figure below:

This picture is the most original one, and we can edit it. For example, the right mouse button can adjust the axis size of PCA diagram. I think the right range of the first axis, that is, the horizontal axis, is too large, which can be appropriately reduced from 2.5 to 2.2.

Get a new picture, as shown below:

We can drag numbers with the mouse, because there are overlapping problems and so on, which is not very pleasant to listen to. Drag it, as shown below, to look better.

In addition, there are several commands with the right mouse button, such as zoom, which can enlarge the atlas and let us see it more clearly. We can also edit the display symbol, text and size of the sample points. For example, in this PCA diagram, samples 1- 12, 1-24 and 25-36 are three treatments respectively. I want to distinguish them intuitively with different symbols. Yes, the operation is as follows: Press Ctrl, select the circular symbol corresponding to the number 13-24, and then press F5 to open the Properties dialog box, where you can edit the color, line, fill, symbol type and size. Of course, as long as you select any object, you can press F5 to edit its features.

The symbol type of the modified sampling point 13-24 is changed as shown in the following figure:

As shown in the figure below, other sampling points can be edited in the same way.

If we are satisfied, we can output pictures. Under the "File" menu, you can choose the format and resolution DPI of the output picture according to your needs. It doesn't matter.