Dependent Software

• fastp
• HISAT2
• samtools
• featureCounts
• stringtie
• AGAT
• TransDecoder

What to input

• Reference genome sequence file
• Reference genome annotation file (in .gtf format)
• RNA-seq fastq files

What to output

Gene expression count matrix.

Usage

1. Prepare your working directory

├── script
│   └── snake_pipeline
├── raw_data
├── genome_index
└── logs

Please storage your resequence data in raw_data/ folder and genome file in genome_index/ folder. Script files, pipeline files and configuration files can be stored in the way you are used to.

2. Prepare the config file

The config file needs to be at the same folder of snakefile.

2.1 Move the genome file to genome_index/ folder and add the genome fasta file absolute path like:

# Absolute path to the genome fasta file
ref: "/workingdir/genome_index/genome.fasta" 

2.2 Sometimes the fastq files may be ended with .fastq.gz or .fq.gz, specify the suffix of the fastq files if it's necessary.

# Fastq file suffix
fastq_suffix: " " # Default value is ".fq.gz"

2.3 Fill in the name of the samples. The samples name need to be filled with specific format like:

# Sample list, samples' name should start with letters.
sample:
    - "sample1"
    - "sample2"
    - "sample3"
    - "sample4"
    - ...
    - "samplen"

You can use following command to add sample list to the config file if you have a sample list txt file (for example sample.list):

# sample.list
sample1
sample2
sample3
sample4

# Add samples to the config file:
awk '{print "    - \"" $0 "\""}' sample.list >> ${working_dir}/RNAseq_config_featureCounts.yaml

3. Submit the pipeline to HPC cluster

For example:

snakemake \
	--snakefile ${snakefile} \
	-d ${working_dir} \
	--cores ${cores_num}