Differential RNA splicing analysis with bulk RNA-seq data

Note

You need to install a Docker image of Shiba (and clone the Shiba GitHub repository to run SnakeShiba). If you don't have them installed, please follow the instructions in the Installation section.

---

Before you start

• Perform mapping of RNA-seq reads to the reference genome and generate bam files by software such as STAR and HISAT2.
• Download a gene annotataion file of your interest in GTF format.

Here is an example code for downloading a mouse gene annotation file (Ensembl 102):

wget https://ftp.ensembl.org/pub/release-102/gtf/mus_musculus/Mus_musculus.GRCm38.102.gtf.gz
gzip -d Mus_musculus.GRCm38.102.gtf.gz

---

Shiba

1. Prepare inputs

experiment.tsv: A tab-separated text file of sample ID, path to bam files, and groups for differential analysis.

sample<tab>bam<tab>group
sample_1<tab>/path/to/workdir/bam/sample_1.bam<tab>Ref
sample_2<tab>/path/to/workdir/bam/sample_2.bam<tab>Ref
sample_3<tab>/path/to/workdir/bam/sample_3.bam<tab>Ref
sample_4<tab>/path/to/workdir/bam/sample_4.bam<tab>Alt
sample_5<tab>/path/to/workdir/bam/sample_5.bam<tab>Alt
sample_6<tab>/path/to/workdir/bam/sample_6.bam<tab>Alt

Please put bam files with their index files (.bai) in the path/to/workdir/bam directory and replace <tab> with a tab character.

config.yaml: A yaml file of the configuration.

workdir:
  /path/to/workdir
gtf:
  /path/to/Mus_musculus.GRCm38.102.gtf
experiment_table:
  /path/to/experiment.tsv
unannotated:
  True

# Junction read filtering
minimum_anchor_length:
  6
minimum_intron_length:
  70
maximum_intron_length:
  500000
strand:
  XS

# PSI calculation
only_psi:
  False
only_psi_group:
  False
fdr:
  0.05
delta_psi:
  0.1
reference_group:
  Ref
alternative_group:
  Alt
minimum_reads:
  10
individual_psi:
  True
ttest:
  True
excel:
  True

You can generate a file of splicing analysis results in excel format by setting excel to True.

2. Run

Docker:

cp experiment.tsv config.yaml /path/to/workdir
cd /path/to/workdir
docker run --rm -v $(pwd):$(pwd) naotokubota/shiba:latest \
python /opt_shiba/Shiba/shiba.py -p 32 /path/to/workdir/config.yaml

Singularity:

cp experiment.tsv config.yaml /path/to/workdir
singularity exec shiba_latest.sif \
python /opt_shiba/Shiba/shiba.py -p 32 /path/to/workdir/config.yaml

Note

When you use Singularity, you do not need to bind any paths as it automatically binds some paths in the host system to the container. In the default configuration, the system default bind points are $HOME, /sys:/sys, /proc:/proc, /tmp:/tmp, /var/tmp:/var/tmp, /etc/resolv.conf:/etc/resolv.conf, /etc/passwd:/etc/passwd, and $PWD. If files needed to be accessed are not in these paths, you can use the --bind option to bind the files to the container.

---

SnakeShiba

A snakemake-based workflow of Shiba. This is useful for running Shiba on a cluster. Snakemake automatically parallelizes the jobs and manages the dependencies between them.

1. Prepare inputs

experiment.tsv: A tab-separated text file of sample ID, path to fastq files, and groups for differential analysis. This is the same as the input for Shiba.

config.yaml: A yaml file of the configuration. This is the same as the configuration for Shiba but with the addition of the container field and without the only_psi and only_psi_group fields as they are not supported in SnakeShiba.

workdir:
  /path/to/workdir
container: # This field is required for SnakeShiba
  docker://naotokubota/shiba
gtf:
  /path/to/Mus_musculus.GRCm38.102.gtf
experiment_table:
  /path/to/experiment.tsv
unannotated:
  True

# Junction read filtering
minimum_anchor_length:
  6
minimum_intron_length:
  70
maximum_intron_length:
  500000
strand:
  XS

# PSI calculation
fdr:
  0.05
delta_psi:
  0.1
reference_group:
  Ref
alternative_group:
  Alt
minimum_reads:
  10
individual_psi:
  True
ttest:
  True
excel:
  True

You can generate a file of splicing analysis results in excel format by setting excel to True.

2. Run

Please make sure that you have installed Snakemake and Singularity and cloned the Shiba repository on your system.

snakemake -s /path/to/Shiba/snakeshiba.smk \
--configfile config.yaml \
--cores 16 \
--use-singularity \
--rerun-incomplete