降解组的分析，目前主流的软件为CleaveLand4，他可以通过测序数据来寻找miRNA与基因的剪切位点，从而验证miRNA靶基因的准确性。

CleaveLand4软件安装

该软件安装非常简单，网址在这，我们只需通过git clone即可对其进行下载。

git clone https://github.com/MikeAxtell/CleaveLand4.git

该软件依赖多个软件，我们需要提前安装bowtie，Vienna RNA，samtools等软件。

CleaveLand4软件使用

通过该软件的提示，我们可以发现他有4种模式。

1. mode1为最常见的模式，输入文件分别为-e 降解组测序的fasta文件，-u miRNA的fasta文件，-n 参考基因组的cds文件，
2. mode2可以作为mode1的后续步骤，当我们需要更换我们的miRNA文件时，我们可以运行mode2，可以省去对测序文件的重新比对，它会直接利用mode1生成的*_dd.txt文件，输入文件只需将-e改为-d *_dd.txt即可。
3. mode3与mode2有类似的效果，当我们的降解组测序文件改变，miRNA文件不变时，我们运行mode3。
4. mode4则是前两个文件都不变，需要做一些条件筛选，或者图片忘记绘制时，它会直接利用mode1生成的文件进行操作，方便我们不用重新跑mode1。

$ CleaveLand4.pl 

CleaveLand4.pl : Finding sliced targets of small RNAs from degradome data

Version: 4.5

Usage: CleaveLand4.pl [options] > [out.txt]

Options:
-h Print help message and quit
-v Print version and quit
-q Quiet mode .. no log/progress information to STDERR
-a Sort small RNA / transcript alignments by Allen et al. score instead of default MFEratio -- for GSTAr
-t Output in tabular format instead of the default verbose format
-r [float >0..1] Minimum Free Energy Ratio cutoff. Default: 0.65 -- for GSTAr
-o [string] : Produce T-plots in the directory indicated by the string. If the dir does not exist, it will be created
-d [string] : Path to degradome density file.
-e [string] : Path to FASTA-formatted degradome reads.
-g [string] : Path to GSTAr-created tabular formatted query-transcript alignments.
-u [string] : Path to FASTA-formatted small RNA queries 
-n [string] : Path to FASTA-formatted transcriptome
-p [float >0..1] : p-value for reporting. Default is 1 (no p-value filtering).
-c [integer 0..4] : Maximum category for reporting. Default is 4 (all categories reported).

Modes:
1. Align degradome data, align small RNA queries, and analyze. 
  REQUIRED OPTIONS: -e, -u, -n
  DISALLOWED OPTIONS: -d, -g
2. Use existing degradome density file, align small RNA queries, and analyze.
  REQUIRED OPTIONS: -d, -u, -n
  DISALLOWED OPTIONS: -e, -g
3. Align degradome data, use existing small RNA query alignments, and analyze.
  REQUIRED OPTIONS: -e, -n, -g
  DISALLOWED OPTIONS: -d, -u
  IRRELEVANT OPTIONS: -a, -r
4. Use existing degradome density file and existing small RNA query alignments, and analyze.
  REQUIRED OPTIONS: -d, -g
  DISALLOWED OPTIONS: -e, -u
  IRRELEVANT OPTIONS: -a, -r

Dependencies (must be in PATH):
  R [only if making T-plots]]
  GSTAr.pl [modes 1 and 2 .. Version 1.0 or higher]
  bowtie (0.12.x OR 1.x) [modes 1, 2, and 3]
  bowtie-build [modes 1, 2, and 3]
  RNAplex (from Vienna RNA Package) [modes 1 and 2]
  samtools [modes 1 and 3]

Documentation: perldoc CleaveLand4.pl

CleaveLand4例子

这里的dga.fasta为二代测序文件fasta转化的fasta格式
-t 输出结果为tab 制表符分割的格式
-p 对输出结果的pvalue进行筛选
-o 每对miRNA与靶基因的剪切位点图片

$ CleaveLand4.pl -e dgd.fasta -u miRNA.fasta -n ref.cds -p 0.05 -t -o test