rMATS进行差异可变剪切分析并可视化

下载：https://www.python.org/
下载后得到文件：Python-2.7.12.tgz，安装过程如下：
tar-zxvf Python-2.7.12.tgz
cd /software/python/Python-2.7.12
./configure--prefix=/software/python/python2712install
make
makeinstall
vim ~/.bashrc
exportPATH="$PATH:/software/python/python2712install/bin";
source ~/.bashrc

下载：https://pypi.org/project/scipy/#files.下载后得到文件numpy-1.16.4.zip，安装过程如下：

unzip numpy-1.16.4.zip
cd numpy-1.16.4/
python2.7 setup.py install

下载：scipy-0.19.0-cp27-cp27m-manylinux1_x86_64.whl
pip2.7 install scipy-0.19.0-cp27-cp27m-manylinux1_x86_64.whl

下载：https://sourceforge.net/projects/samtools/files/

tar -xjf samtools-1.3.1.tar.bz2
cd samtools-1.3.1/
./configure--prefix=/software/samtools/samtools-1.3.1-install
make
makeinstall
vim ~/.bashrc
exportPATH="$PATH:/software/samtools/samtools-1.3.1-install/bin";
source ~/.bashrc

下载：https://github.com/alexdobin/STAR

unzip STAR-master.zip
vim ~/.bashrc
exportPATH="$PATH:/software/STAR/STAR-master/bin/Linux_x86_64";
source ~/.bashrc

下载：http://rnaseq-mats.sourceforge.net/

cd /software/rMATS
tar -zxcf /software/rMATS
cd rMATS.4.0.2

>>>import sys
>>>print sys.maxunicode
1114111
Thisoutput indicates that your python is built with --enable-unicode=ucs4, and youshould use rMATS-turbo-xxx-UCS4.
>>>import sys
>>>print sys.maxunicode
65535

python/software/rMATS/rMATS.4.0.2/rMATS-turbo-Linux-UCS4/rmats.py --help

下载：https://github.com/Xinglab/rmats2sashimiplot/

unzip rmats2sashimiplot-master.zip
cd rmats2sashimiplot-master/
python2.7 setup.py install

cat  s1.txt
231ESRP.25K.rep-1.R1.fastq:231ESRP.25K.rep-1.R2.fastq,231ESRP.25K.rep-2.R1.fastq:231ESRP.25K.rep-2.R2.fastq

cat  s2.txt
231EV.25K.rep-1.R1.fastq:231EV.25K.rep-1.R2.fastq,231EV.25K.rep-2.R1.fastq:231EV.25K.rep-2.R2.fastq

python2.7 /software/rMATS/rMATS.4.0.2/rMATS-turbo-Linux-UCS4/rmats.py --s1 s1.txt --s2 s2.txt --gtf Homo_sapiens.GRCh37.75.gtf --bi STARindexFolder -od outDir -tpaired --readLength 50 --libType fr-unstranded --nthread 10

python2.7 /software/rMATS/rMATS.4.0.2/rMATS-turbo-Linux-UCS4/rmats.py --b1 b1.txt --b2 b2.txt --gtf Homo_sapiens.GRCh37.75.gtf --od outDir -t paired --readLength 50--libType fr-unstranded --nthread 10

--s1 s1.txt  A text file contains FASTQfile(s) for the sample_1.文件内以逗号分隔重复样本的FASTQ文件名(Onlyif using fastq)
--s2 s2.txt  A text file contains FASTQfile(s) for the sample_2.文件内以逗号分隔重复样本的FASTQ文件名(Onlyif using fastq)
--b1 b1.txt  A text file records mappingresults for the sample_1 in bam format. 文件内以逗号分隔重复样本的bam文件名(Only if using bam)
--b2 b2.txt  A text file records mappingresults for the sample_2 in bam format. 文件内以逗号分隔重复样本的bam文件名(Only if using bam)
-treadType  Type of read used in theanalysis. readType is either 'paired' or 'single'. 'paired' is for paired-enddata and 'single' is for single-end data
--readLength  测序reads的长度
--gtf  gtfFile 需要输入的gtf格式文件
--bi  STARIndexFolderThe folder name of the STARbinary indexes (i.e., the name of the folder that contains SA file). Forexample, use ~/STARindex/hg19 for hg19. (Only if using fastq)
--odoutDir  所有输出文件的路径
--tophatAnchorThe "anchor length" or "overhang length" used in thealigner. At least “anchor length” NT must be mapped to each end of a givenjunction. The default is 6. (This parameter applies only if using fastq)
--nthreadThe number of thread.
--cstat  The cutoff splicing difference. The cutoffused in the null hypothesis test for differential splicing. The default is0.0001 for 0.01% difference. Valid: 0 ≤ cutoff < 1
--tstat  The number of thread for statistical model.
--statoff  Turn statistics part off.
-libTypelibraryTypeLibrary type.  Default isunstranded (fr-unstranded). Use fr-firststrand or fr-secondstrand forstrand-specific data.

1）ID
2）GeneID
3）geneSymbol
4）chr
5）strand
6）exonStart_0base
7）exonEnd
8）upstreamES
9）upstreamEE
10）downstreamES
11）downstreamEE
12）ID
13）IJC_SAMPLE_1：样品在inclusion junction下的count数，重复样本的结果以逗号分隔
14）SJC_SAMPLE_1：样品在skipping junction counts（SJC）下的count数，重复样本的结果以逗号分隔
15）IJC_SAMPLE_2：样品在inclusion junction下的count数，重复样本的结果以逗号分隔
16）SJC_SAMPLE_2：样品在skipping junction counts（SJC）下的count数，重复样本的结果以逗号分隔
17）IncFormLen：lengthof inclusion form, used for normalization
18）SkipFormLen：lengthof skipping form, used for normalization
19）PValue：Significanceof splicing difference between two sample groups（两组样品可变剪切的统计学显著差异指标）
20）FDR：FalseDiscovery Rate calculated from p-value（校正后的PValue）
21）IncLevel1：inclusionlevel for SAMPLE_1 replicates (comma separated) calculated from normalizedcounts
22）IncLevel2：inclusionlevel for SAMPLE_2 replicates (comma separated) calculated from normalizedcounts
23）IncLevelDifference：average(IncLevel1)– average(IncLevel2)

rmats2sashimiplot --b1 231ESRP.25K.rep-1.bam,231ESRP.25K.rep-2.bam --b2 231EV.25K.rep-1.bam,231EV.25K.rep-2.bam -t SE -e /software/rMATS/test/outDir/SE.MATS.JC.txt --l1 231ESRP.25K --l2 231EV.25K -o SE_plot

--b1 B1 sample_1 in bam format(s1_rep1.bam[,s1_rep2.bam])
--b2 B2 sample_2 in bam format(s2_rep1.bam[,s2_rep2.bam])
-t  rMATS结果中产生的可变剪切类型{SE,A5SS,A3SS,MXE,RI}
-e  EVENTS_FILE The rMATS output event file (Onlyif using rMATSformat result as event file).
--l1 L1 The label for first sample.
--l2 L2 The label for second sample.
-o OUT_DIR The output directory.