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。
在这里插入图片描述
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。
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，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。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。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
