/mnt/smb/notebooktian/RNASeq/260429AGING

### References
 * MAJIQ [RNA splicing analysis using heterogeneous and large RNA-seq datasets - nature communications](https://www.nature.com/articles/s41467-023-36585-y)
 * MAJIQ -> cryptic 3'splice site
 
 * A3SS 图B， 有拖尾感的sashimi [Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3′ Splice Site Selection through Use of a Different Branch Point](https://www.cell.com/cell-reports/fulltext/S2211-1247(15)01078-5)


### MAJIQ

https://bitbucket.org/biociphers/majiq_academic/src/main/

https://biociphers.bitbucket.io/majiq-docs/

```dockerfile
FROM python:3.12-slim-bookworm

ENV DEBIAN_FRONTEND=noninteractive

# 1. 安装系统依赖（已增加 libzstd-dev）
RUN apt-get update && apt-get install -y --no-install-recommends \
    build-essential \
    git \
    libhts-dev \
    libbz2-dev \
    zlib1g-dev \
    liblzma-dev \
    libzstd-dev \
    && rm -rf /var/lib/apt/lists/*

ENV HTSLIB_LIBRARY_DIR=/usr/lib/x86_64-linux-gnu
ENV HTSLIB_INCLUDE_DIR=/usr/include

# 2. 拆分指令以利用缓存 (存档点：下载源码)
WORKDIR /tmp
RUN git clone https://bitbucket.org/biociphers/majiq_academic.git

# 3. 存档点：安装 Voila
WORKDIR /tmp/majiq_academic
RUN pip install --no-cache-dir ./moccasin
RUN pip install --no-cache-dir ./voila

# 4. 存档点：安装 MAJIQ
RUN pip install --no-cache-dir ./majiq

WORKDIR /
RUN rm -rf /tmp/majiq_academic

RUN majiq --help && voila --version

CMD ["/bin/bash"]
```

#### License
 *  放入 ~/majiq_license_academic_official.lic
```majiq_license_academic_official.lic
gAAAAABlW6-jYooOgeYvzqoEZ9Z6jMLcFAfydEzRxJWwJ7bl9z71xmQCS343USkuLpu88lpTwTOGiGqpJ0uMZV36nto1SCRmdP_LWSugZyS7-E1wDVOzyV8kQcUHzNtPQVMVaLUQXmpYGfXGHEVo-XUPVG3v3waurw==
```


### Steps v3
```bash
alias majiq='docker run --rm -e MAJIQ_LICENSE_FILE=/data/majiq_license_academic_official.lic -v "$(pwd)/datamj:/data" -w /data majiq_image majiq'
alias voila='docker run --rm -e MAJIQ_LICENSE_FILE=/data/majiq_license_academic_official.lic -v "$(pwd)/datamj:/data" -p 5000:5000 -w /data majiq_image voila'
alias majiq3='docker run --rm -e MAJIQ_LICENSE_FILE=/data/majiq_license_academic_official.lic -v "$(pwd)/datamj:/data" -w /data majiq_image majiq-v3'

PATH_GFF3="/data/mm39_115.gff3"
PATH_BAMS="/data/bams"
PATH_ANNO_SG="/data/mj/anno_sg.zarr"
PATH_SJ="/data/mj/sjs"
PATH_SG_ZAAR="/data/mj/sg.zarr"
PATH_PSICOV="/data/mj/psi"
PATH_SGC="/data/mj/sgc"
PATH_VOILA="/data/mj/voila"


majiq3 gff3 $PATH_GFF3 $PATH_ANNO_SG
majiq3 sj $PATH_BAMS/OLDSED_1.bam $PATH_ANNO_SG $PATH_SJ/OLDSED_1.sj
majiq3 sj $PATH_BAMS/OLDSED_2.bam $PATH_ANNO_SG $PATH_SJ/OLDSED_2.sj
majiq3 sj $PATH_BAMS/OLDSED_3.bam $PATH_ANNO_SG $PATH_SJ/OLDSED_3.sj
majiq3 sj $PATH_BAMS/YNGSED_1.bam $PATH_ANNO_SG $PATH_SJ/YNGSED_1.sj
majiq3 sj $PATH_BAMS/YNGSED_2.bam $PATH_ANNO_SG $PATH_SJ/YNGSED_2.sj
majiq3 sj $PATH_BAMS/YNGSED_3.bam $PATH_ANNO_SG $PATH_SJ/YNGSED_3.sj

majiq3 build $PATH_ANNO_SG $PATH_SG_ZAAR --sjs $PATH_SJ/OLDSED_1.sj $PATH_SJ/OLDSED_2.sj $PATH_SJ/OLDSED_3.sj --sjs $PATH_SJ/YNGSED_1.sj $PATH_SJ/YNGSED_2.sj $PATH_SJ/YNGSED_3.sj

majiq3 psi-coverage $PATH_SG_ZAAR $PATH_PSICOV/OLDSED.psicov $PATH_SJ/OLDSED_1.sj $PATH_SJ/OLDSED_2.sj $PATH_SJ/OLDSED_3.sj
majiq3 psi-coverage $PATH_SG_ZAAR $PATH_PSICOV/YNGSED.psicov $PATH_SJ/YNGSED_1.sj $PATH_SJ/YNGSED_2.sj $PATH_SJ/YNGSED_3.sj

majiq3 sg-coverage $PATH_SG_ZAAR $PATH_SGC/OLDSED.sgc $PATH_SJ/OLDSED_1.sj $PATH_SJ/OLDSED_2.sj $PATH_SJ/OLDSED_3.sj
majiq3 sg-coverage $PATH_SG_ZAAR $PATH_SGC/YNGSED.sgc $PATH_SJ/YNGSED_1.sj $PATH_SJ/YNGSED_2.sj $PATH_SJ/YNGSED_3.sj


majiq3 deltapsi --splicegraph $PATH_SG_ZAAR --output-voila $PATH_PSICOV/OLDSED_v_YNGSED.psicov --output-tsv $PATH_PSICOV/OLDSED_v_YNGSED.tsv -psi1 $PATH_PSICOV/OLDSED.psicov -psi2 $PATH_PSICOV/YNGSED.psicov

majiq3 quantify $PATH_PSICOV/OLDSED.psicov --min-experiments 0.01 --splicegraph $PATH_SG_ZAAR --output-tsv $PATH_PSICOV/OLDSED.tsv --overwrite
majiq3 quantify $PATH_PSICOV/YNGSED.psicov --min-experiments 0.01 --splicegraph $PATH_SG_ZAAR --output-tsv $PATH_PSICOV/YNGSED.tsv --overwrite

majiq3 heterogen --stats infoscore mannwhitneyu ttest tnom --splicegraph $PATH_SG_ZAAR --output-voila $PATH_VOILA/OLDSED_v_YNGSED.voila --output-tsv $PATH_PSICOV/OLDSED_v_YNGSED.tsv -psi1 $PATH_PSICOV/OLDSED.psicov -psi2 $PATH_PSICOV/YNGSED.psicov




voila view --host 0.0.0.0 --port 5000 --enable-type-indexing --psicov-grouping-file /data/majiq.tsv $PATH_SG_ZAAR $PATH_VOILA/OLDSED_v_YNGSED.voila $PATH_SGC/

```

### Steps v2
```bash
majiq build /data/mm39_115.gff3 -c /data/majiq.config -o /data/build_output
majiq psi-coverage \
    /data/build_output/splicegraph.zarr \
    /data/psi_coverage_output \
    /data/build_output/YNGSED_1.sj /data/build_output/YNGSED_2.sj /data/build_output/YNGSED_3.sj \
    /data/build_output/OLDSED_1.sj /data/build_output/OLDSED_2.sj /data/build_output/OLDSED_3.sj \
    --nthreads 8

majiq deltapsi \
    -grp1 /data/psi_coverage_output \
    -grp2 /data/psi_coverage_output \
    --select-grp1-prefixes YNGSED_1 YNGSED_2 YNGSED_3 \
    --select-grp2-prefixes OLDSED_1 OLDSED_2 OLDSED_3 \
    --names YNGSED OLDSED \
    --splicegraph /data/build_output/splicegraph.zarr \
    --output-voila /data/deltapsi_output/YNGSED_vs_OLDSED.voila \
    --output-tsv /data/deltapsi_output/YNGSED_vs_OLDSED.tsv \
    --nthreads 8

majiq sg-coverage /data/build_output/splicegraph.zarr /sgc/YNGSED.sgc /data/build_output/YNGSED_1.sj /data/build_output/YNGSED_2.sj /data/build_output/YNGSED_3.sj


voila view /data/build_output/ --host 0.0.0.0 --port 5000 --enable-type-indexing --psicov-grouping-file /data/majiq.tsv

```