安装

  1. 要求

    • 32G内存或者更高 (根据参考基因组大小确定)
    • Linux
    • conda环境管理器
  2. 使用conda安装

    • 创建环境

      conda create -n scope
      
    • 激活环境

      conda activate scope
      
    • 安装scopetools

      conda install -c singleronbio scope-tools
      
    • 验证安装完成

      scope -h
      
      Usage: scope [OPTIONS] COMMAND1 [ARGS]... [COMMAND2 [ARGS]...]...
      
        Single Cell Omics Preparation Entity Tools
      
      Options:
        --version   Show the version and exit.
        -h, --help  Show this message and exit.
      
      Commands:
        STAR           STAR short help
        barcode        extract barcode and umi short help
        cluster        cluster short help
        count          count short help
        cutadapt       cutadapt short help
        featureCounts  featureCounts short help
        run            run short help
      
  3. 准备参照基因组

    • Homo sapiens

      wget ftp://ftp.ensembl.org/pub/release-99/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
      wget ftp://ftp.ensembl.org/pub/release-99/gtf/homo_sapiens/Homo_sapiens.GRCh38.99.gtf.gz
      
      mkdir -p references/Homo_sapiens/Ensembl/GRCh38
      gzip -c -d Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz > references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.fa
      gzip -c -d Homo_sapiens.GRCh38.99.gtf.gz > references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.gtf
      
      conda activate scope
      
      gtfToGenePred -genePredExt -geneNameAsName2 references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.gtf /dev/stdout | \
          awk '{print $12"\t"$1"\t"$2"\t"$3"\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9"\t"$10}' > references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.refFlat
      
      STAR \
          --runMode genomeGenerate \
          --runThreadN 6 \
          --genomeDir references/Homo_sapiens/Ensembl/GRCh38 \
          --genomeFastaFiles references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.fa \
          --sjdbGTFfile references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.gtf \
          --sjdbOverhang 100
      
    • Mus musculus

      wget ftp://ftp.ensembl.org/pub/release-99/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
      wget ftp://ftp.ensembl.org/pub/release-99/gtf/mus_musculus/Mus_musculus.GRCm38.99.gtf.gz
      
      mkdir -p references/Mus_musculus/Ensembl/GRCm38
      gzip -c -d Mus_musculus.GRCm38.dna.primary_assembly.fa.gz > references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.fa
      gzip -c -d Mus_musculus.GRCm38.99.gtf.gz > references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.gtf
      
      conda activate scope
      
      gtfToGenePred -genePredExt -geneNameAsName2 references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.gtf /dev/stdout | \
          awk '{print $12"\t"$1"\t"$2"\t"$3"\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9"\t"$10}' > references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.refFlat
      
      STAR \
          --runMode genomeGenerate \
          --runThreadN 6 \
          --genomeDir references/Mus_musculus/Ensembl/GRCm38 \
          --genomeFastaFiles references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.fa \
          --sjdbGTFfile references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.gtf \
          --sjdbOverhang 100