skipped exon (SE)，外显子跳跃，指一个或多个外显子连同其两端的内含子一起被剪切，在成熟mRNA中不存在。

alternative 5' splice site (A5SS)，5’端可变剪接，它们的3’端剪接位点一致但5’端剪接位点不同，产生不同长度的5’端外显子。

alternative 5' splice site (A3SS)，3’端可变剪接，它们的5’端剪接位点一致但3’端剪接位点不同，产生不同长度的3’端外显子。

mutually exclusive exons (MXE)，外显子互斥，成熟的mRNA变体中，彼此特有的外显子，这些外显子不能同时出现在同一成熟mRNA中。

retained intron (RI)，内含子保留，在一些转录本中内含子不会被剪切掉，保留在成熟的mRNA。

定量

rMATS采用exon inclusion level 来定义样本中可变剪切事件的表达量，以外显子跳跃（Skipped Exon）为例，正常的转录本称之为Exon Inclusion Isofrom, 发生了外显子跳跃的转录本则称之为Exon Skipping Isofrom。

用 I 表示比对到Exon Inclusion Isofrom上的reads，S表示比对到Exon Skipping Isofrom上的reads， 则该外显子跳跃的可变剪切事件比例可以表示为：

可以看到，exon inclusion level实际上是inclusion isofrom所占的比例，计算时，用长度校正了原始的reads数。其他类型的可变剪切事件也可以划分成上述两种isoform, 示意图如下:

可以看到，rmats在计算isofrom的长度时，提供了两种方式，二者的区别就在于是否考虑跳过的exon的长度。

conda create -n my_rmats_env
conda activate my_rmats_env
conda install rmats-turbo
rmats-turbo --version

python rmats.py -h
usage: rmats.py [options]
optional arguments:
 -h, --help            show this help message and exit
 --version             show program's version number and exit
  --gtf GTF             An annotation of genes and transcripts in GTF format
 --b1 B1               A text file containing a comma separated list of the
                        BAM files for sample_1. (Only if using BAM)
 --b2 B2               A text file containing a comma separated list of the
                        BAM files for sample_2. (Only if using BAM)
  --s1 S1               A text file containing a comma separated list of the
                        FASTQ files for sample_1. If using paired reads the
                        format is ":" to separate pairs and "," to separate
                        replicates. (Only if using fastq)
  --s2 S2               A text file containing a comma separated list of the
                        FASTQ files for sample_2. If using paired reads the
                        format is ":" to separate pairs and "," to separate
                        replicates. (Only if using fastq)
  --od OD               The directory for final output
  --tmp TMP             The directory for intermediate output such as ".rmats"
                        files from the prep step
  -t {paired,single}    Type of read used in the analysis: either "paired" for
                        paired-end data or "single" for single-end data.
                        Default: paired
  --libType {fr-unstranded,fr-firststrand,fr-secondstrand}
                        Library type. Use fr-firststrand or fr-secondstrand
                        for strand-specific data. Default: fr-unstranded
  --readLength READLENGTH
                        The length of each read
  --variable-read-length
                        Allow reads with lengths that differ from --readLength
                        to be processed. --readLength will still be used to
                        determine IncFormLen and SkipFormLen
  --anchorLength ANCHORLENGTH
                        The anchor length. Default is 1
  --tophatAnchor TOPHATANCHOR
                        The "anchor length" or "overhang length" used in the
                        aligner. At least "anchor length" NT must be mapped to
                        each end of a given junction. The default is 6. (Only
                        if using fastq)
  --bi BINDEX           The directory name of the STAR binary indices (name of
                        the directory that contains the SA file). (Only if
                        using fastq)
  --nthread NTHREAD     The number of threads. The optimal number of threads
                        should be equal to the number of CPU cores. Default: 1
  --tstat TSTAT         The number of threads for the statistical model.
                        Default: 1
  --cstat CSTAT         The cutoff splicing difference. The cutoff used in the
                        null hypothesis test for differential splicing. The
                        default is 0.0001 for 0.01% difference. Valid: 0 <=
                        cutoff < 1. Does not apply to the paired stats model
  --task {prep,post,both,inte}
                        Specify which step(s) of rMATS to run. Default: both.
                        prep: preprocess BAMs and generate a .rmats file.
                        post: load .rmats file(s) into memory, detect and
                        count alternative splicing events, and calculate P
                        value (if not --statoff). both: prep + post. inte
                        (integrity): check that the BAM filenames recorded by
                        the prep task(s) match the BAM filenames for the
                        current command line
  --statoff             Skip the statistical analysis
  --paired-stats        Use the paired stats model
  --novelSS             Enable detection of novel splice sites (unannotated
                        splice sites). Default is no detection of novel splice
                        sites
  --mil MIL             Minimum Intron Length. Only impacts --novelSS
                        behavior. Default: 50
  --mel MEL             Maximum Exon Length. Only impacts --novelSS behavior.
                        Default: 500

##/path/to/b1.txt
/path/to/1_1.bam,/path/to/1_2.bam
##/path/to/b2.txt
/path/to/2_1.bam,/path/to/2_2.bam
python rmats.py --b1 /path/to/b1.txt --b2 /path/to/b2.txt --gtf Gallus_gallus.GRCg6a.101.gtf --od A_vs_B --tmp A_vs_B/tmp -t paired --variable-read-length --readLength 150 --cstat 0.0001  --libType fr-unstranded  --novelSS --nthread 4