Gepard Linux Command line mode

  Gepard软件能快速对两个fasta格式的序列进行共线性分析，获得二维点图。我利用该软件判断由GetOrganelle组装获得的叶绿体ssc区的方向。尽管这是2007年就发布的软件^[1]，但应付上述目的绰绰有余。

图1 Gepard在windows中的可视化软件界面，这里是对Cas_hy, cas004-2两个叶绿体基因组文件进行分析

  之前已经尝试过windows的版本（参考^[3]），但当有大量组装完成的叶绿体ssc需要判断时，就有必要批处理了。Gepard的linux安装利用conda或Docker，相当便捷，参考https://github.com/univieCUBE/gepard。

  安装后键入Gepardcmd弹出help信息

Gepard 2.0 - command line mode

Reference:
Krumsiek J, Arnold R, Rattei T
Gepard: A rapid and sensitive tool for creating dotplots on genome scale.
Bioinformatics 2007; 23(8): 1026-8. PMID: 17309896

Parameters are supplied as -name value

Required parameters:
  -seq:        the sequences, seperated by spaces. The first gets paired to the second, third to fourth and so on.
  -matrix:      substitution matrix file
  -outfile:     output file name

... (Following omission)

  -seq, -matrix, -outfile是必需的，-seq, -outfile很好理解，与windows界面不同的是需要键入-matrix，这是要求输入一个核苷酸替代矩阵，官方tutorial推荐matrices/edna.mat。conda安装的通过以下命令找到这个替代矩阵：

$ which gepard
~/miniconda3/envs/gepard/bin/gepard
$ cd ~/miniconda3/envs/gepard
$ find -name edna.mat
./share/gepard/resources/matrices/edna.mat
./share/gepard/src/matrices/edna.mat
$ cd ./share/gepard/resources/matrices
$ less edna.mat
#
# This matrix was created by Todd Lowe 12/10/92
#
# Uses ambiguous nucleotide codes, probabilities rounded to
# nearest integer
#
# Lowest score = -4, Highest score = 5
#
# modified for use with gepard (delimiter letter Z)

   A  T  G  C  N  W  R  Y  K  M  B  V  H  D  S  U  Z  X 
A  1  0  0  0 -2 -4  1  1 -4 -4  1 -4 -1 -1 -1 -4 -9 -9
T  0  1  0  0 -2 -4  1 -4  1  1 -4 -1 -4 -1 -1  5 -9 -9
G  0  0  1  0 -2  1 -4  1 -4  1 -4 -1 -1 -4 -1 -4 -9 -9
C  0  0  0  1 -2  1 -4 -4  1 -4  1 -1 -1 -1 -4 -4 -9 -9
N -2 -2 -2 -2 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -2 -9 -9
W -4 -4  1  1 -1 -1 -4 -2 -2 -2 -2 -1 -1 -3 -3 -4 -9 -9
R  1  1 -4 -4 -1 -4 -1 -2 -2 -2 -2 -3 -3 -1 -1  1 -9 -9
Y  1 -4  1 -4 -1 -2 -2 -1 -4 -2 -2 -3 -1 -3 -1 -4 -9 -9
K -4  1 -4  1 -1 -2 -2 -4 -1 -2 -2 -1 -3 -1 -3 -1 -9 -9
M -4  1  1 -4 -1 -2 -2 -2 -2 -1 -4 -1 -3 -3 -1  1 -9 -9
B  1 -4 -4  1 -1 -2 -2 -2 -2 -4 -1 -3 -1 -1 -3 -4 -9 -9
V -4 -1 -1 -1 -1 -1 -3 -3 -1 -1 -3 -1 -2 -2 -2 -1 -9 -9
H -1 -4 -1 -1 -1 -1 -3 -1 -3 -3 -1 -2 -1 -2 -2 -4 -9 -9
D -1 -1 -4 -1 -1 -3 -1 -3 -1 -3 -1 -2 -2 -1 -2 -1 -9 -9
S -1 -1 -1 -4 -1 -3 -1 -1 -3 -1 -3 -2 -2 -2 -1 -1 -9 -9
U -4  5 -4 -4 -2 -4  1 -4  1  1 -4 -1 -4 -1 -1  5 -9 -9
Z -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9
X -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9 -9

  知道矩阵存放位置后，还需找到软件存放地址（因为我试验环境变量的命令直接调用跑不通，不清楚是为什么），实际上就是.../share/gepard下dist，里面有Gepard-1.40.jar Gepard-2.1.jar两个文件，使用Gepard-2.1.jar（两个程序都可用，但命令略有不同，以后者为例）。

  然后就可以运行软件了。由于官方tutorial文件没有及时更新，实际的运行命令应该是（对应Gepard-2.1版本，即现在下载默认的版本）^[2]：

java -cp ~/miniconda3/envs/gepard/share/gepard/dist/Gepard-2.1.jar org.gepard.client.cmdline.CommandLine \
-seq ref.fasta test.fasta \
-matrix ~/miniconda3/envs/gepard/share/gepard/resources/matrices/edna.mat \
-outfile test1.png

  运行会报错并弹出一个窗口，需要安装xmanager 11什么的，就按指示安装，第一次安装有个30天试用，管他呢，先用了，后面用到再想办法...：

Loading substitution matrix...
Loading sequence from ref.fasta
Loading sequence from test.fasta
Calculating suffix array... 
Calculating dotplot... 
Creating image and writing to file... 
Exception in thread "main" java.awt.AWTError: Can't connect to X11 window server using 'localhost:12.0' as the value of the DISPLAY variable.
    at java.desktop/sun.awt.X11GraphicsEnvironment.initDisplay(Native Method)
    at java.desktop/sun.awt.X11GraphicsEnvironment$1.run(X11GraphicsEnvironment.java:104)
    at java.base/java.security.AccessController.doPrivileged(Native Method)
    at java.desktop/sun.awt.X11GraphicsEnvironment.<clinit>(X11GraphicsEnvironment.java:63)
    at java.base/java.lang.Class.forName0(Native Method)
    at java.base/java.lang.Class.forName(Class.java:315)
    at java.desktop/java.awt.GraphicsEnvironment$LocalGE.createGE(GraphicsEnvironment.java:101)
    at java.desktop/java.awt.GraphicsEnvironment$LocalGE.<clinit>(GraphicsEnvironment.java:83)
    at java.desktop/java.awt.GraphicsEnvironment.getLocalGraphicsEnvironment(GraphicsEnvironment.java:129)
    at java.desktop/java.awt.image.BufferedImage.createGraphics(BufferedImage.java:1181)
    at java.desktop/java.awt.image.BufferedImage.getGraphics(BufferedImage.java:1170)
    at org.gepard.client.Plotter.<init>(Plotter.java:92)
    at org.gepard.client.cmdline.CommandLine.main(CommandLine.java:304)

  安装完再运行，就正常，

Loading substitution matrix...
Loading sequence from ref.fasta
Loading sequence from test.fasta
Calculating suffix array... 
Calculating dotplot... 
Creating image and writing to file...

  最后写个循环就实现批处理啦！

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参考资料：
[1] Krumsiek J, Arnold R, Rattei T. Gepard: a rapid and sensitive tool for creating dotplots on genome scale[J]. Bioinformatics, 2007, 23(8): 1026-1028.
[2] How to start gepard on the commandline.
[3] 被子植物·叶绿体组装、注释与比较分析·框架