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Notes

Repo Files and Directories

  • scripts/: containing bash and SLURM scripts for scRNA analysis, database download, and other utilities
    • download_refgenome.sh: script for downloading reference genomes, transcriptomes, and GTFs
    • download_sra.sbatch: SLURM script to download SRA samples from NCBI
    • fastqc.sbatch: SLURM script to run FastQC
    • star_index.sh: bash script to index reference genome with STAR
    • star_align.sbatch: SLURM script to map reads to the indexed reference genome and run STARSolo

Other files in the repository:

  • setup.sh: bash script to set up container and default paths
  • Dockerfile: Docker image to build container
  • README.md: project README
  • Notes.md: running documentation

Usage

  • download_refgenome.sh

    ./download_refgenome.sh <output_directory_path>

  • download_sra.sbatch

    sbatch download_sra.sh SRP SRP266243 $RAW_DATA/sra

  • fastqc.sbatch

    sbatch fastqc.sbatch $RAW_DATA/sra/SRP266243/SRR11940660 \
                     $PROCESSED_DATA/SRP266243/SRR11940660/fastqc_outs

  • star_index.sh

    ./star_index.sh <input_fasta> <input_gtf> <output_folder>
# Example:
./star_index.sh $ECOLI_REF_GENOME $ECOLI_GTF \
                $PROCESSED_DATA/ref_index/STAR_ecoli_genome_index

  • star_align.sbatch

    # Usage:
sbatch star_align.sbatch \
       $PROCESSED_DATA/ref_index/STAR_bsub_genome_index \
       $RAW_DATA/sra/SRP266243/SRR11940660/SRR11940660_1.fastq \
       $RAW_DATA/sra/SRP266243/SRR11940660/SRR11940660_2.fastq \
       $RAW_DATA/barcodes/SRP266243/barcodes_r2_r3_solo.txt \
       $RAW_DATA/barcodes/SRP266243/barcodes_r1_solo.txt \
       $PROCESSED_DATA/SRP266243/SRR11940660/STAR_Bsub_ref_outs

Data Organization

scRNAseq/

  • envs/: directory containing environment files

    • scrna_latest.sif: singularity image file

  • processed_data/ ($PROCESSED_DATA): contains analysis outputs

    • ref_index/: indexed reference genomes
      • STAR_alt_bsub_genome_index: star_index.sh output for reference genome of B.subtilis PY79
      • STAR_bsub_genome_index: star_index.sh output for reference genome of B.subtilis strain 168
      • STAR_ecoli_genome_index: star_index.sh output for reference genome of E.coli K-12 substr. MG1655
    • SRP266243/: outputs for SRP266243
      • SRR11940660/: outputs for SRR11940660 runs
        • fastqc_outs: fastqc.sbatch output
        • STAR_alt_bsub_ref_outs: STAR outputs (aligning reads to STAR_alt_bsub_genome_index)
        • STAR_Bsub_ref_outs: STAR outputs (aligning reads to STAR_bsub_genome_index)

  • raw_data/ ($RAW_DATA): containing raw data files

    • barcodes/: barcodes from Supplementary information from this paper
      • SRP266243/
    • references/
      • bsubtilis: reference genomes/transcriptomes/gtf files of B.subtilis strain 168 and PY79
      • ecoli: reference genomes/transcriptomes/gtf files of E.coli K-12 substr. MG1655
    • sra/
      • SRP266243/: this project has 3 runs
        • SRR11940660: B.subtilis rep 1
        • SRR11940661: B.subtilis rep 2
        • SRR11940662: B.subtilis + E.coli

Methods

  1. Run setup.sh script to set up environment variables and singularity run

  2. Prepare necessary files in $RAW_DATA

Download the reference files:

./download_refgenome.sh $RAW_DATA

Download SRA reads:

sbatch download_sra.sh SRP SRP266243 $RAW_DATA/sra

Copy barcodes from Supplementary information to text files in $RAW_DATA/barcodes

  1. Convert file formats
gffread $BSUB_PY79_GFF -g $BSUB_PY79_REF_GENOME -T -v -o $BSUB_PY79_GTF
  1. Run fastqc.sbatch on SRR11940660 (B. subtilis experiment):
sbatch fastqc.sbatch $RAW_DATA/sra/SRP266243/SRR11940660 \
                     $PROCESSED_DATA/SRP266243/SRR11940660/fastqc_outs
  1. Index reference genomes

E.coli:

./star_index.sh $ECOLI_REF_GENOME $ECOLI_GTF \
                $PROCESSED_DATA/ref_index/STAR_ecoli_genome_index

B.subtilis strain 168:

./star_index.sh $BSUB_REF_GENOME $BSUB_GTF \
                $PROCESSED_DATA/ref_index/STAR_bsub_genome_index

B.subtilis strain PY79:

./star_index.sh $BSUB_PY79_REF_GENOME $BSUB_PY79_GTF \
                $PROCESSED_DATA/ref_index/STAR_alt_bsub_genome_index
  1. STARsolo align

B.subtilis strain 168:

sbatch star_align.sbatch \
       $PROCESSED_DATA/ref_index/STAR_bsub_genome_index \
       $RAW_DATA/sra/SRP266243/SRR11940660/SRR11940660_1.fastq \
       $RAW_DATA/sra/SRP266243/SRR11940660/SRR11940660_2.fastq \
       $RAW_DATA/barcodes/SRP266243/barcodes_r2_r3_solo.txt \
       $RAW_DATA/barcodes/SRP266243/barcodes_r1_solo.txt \
       $PROCESSED_DATA/SRP266243/SRR11940660/STAR_Bsub_ref_outs

B.subtilis strain PY79:

sbatch star_align.sbatch \
       $PROCESSED_DATA/ref_index/STAR_alt_bsub_genome_index \
       $RAW_DATA/sra/SRP266243/SRR11940660/SRR11940660_1.fastq \
       $RAW_DATA/sra/SRP266243/SRR11940660/SRR11940660_2.fastq \
       $RAW_DATA/barcodes/SRP266243/barcodes_r2_r3_solo.txt \
       $RAW_DATA/barcodes/SRP266243/barcodes_r1_solo.txt \
       $PROCESSED_DATA/SRP266243/SRR11940660/STAR_alt_bsub_ref_outs