APPRIS RNA-Seq pipeline

• APPRIS RNA-Seq pipeline
  • Introduction
  • Installation
  • Usage
  • Directory structure
  • Author information and license
  • Release History
  • Contributing

Introduction

The Snakemake pipeline presented here allow to align a batch of RNA-seq paired-end samples accounting for genomic features.

It has been thought to be runned in a High-performance computing environment, but it could be adapted depending on the computer capability.

It allows both GENCODE and RefSeq genome reference annotations.

Sequentially it executes:

• Cutadapt: To find and removes adapters. It uses the cutadapt_pe mode.
• STAR: Spliced and referenced transcripts aligner.
• Samtools: To interact with the sequencing data.
• featureCounts: To count reads to genomic features such as genes or exons.

QSplice could also be runned after the pipeline in order to quantify the splice junctions coverage per transcript.

Installation

Run the silent installation of Miniconda in case you don't have this software in your Linux Environment

wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
bash Miniconda3-latest-Linux-x86_64.sh -b -p $HOME/miniconda3

Once you have installed Miniconda/Anaconda, create a Python 3.7 environment. Then, install snakemake in your conda environment:

conda install -c conda-forge mamba
mamba create -c conda-forge -c bioconda -n snakemake snakemake

If Slurm has been installed in the computing environment, install with:

sbatch -o log.txt -e err.txt -J smk-install -c 2 --mem=2G -t 200 --wrap "mamba create -c conda-forge -c bioconda -y -n snakemake snakemake"

Usage

To execute the pipeline, first the user must prepare their samples.

git clone git@gitlab.com:fpozoc/appris_rnaseq.git
cd appris_rnaseq

Then, edit config and workflow as needed:

vim config/config.yaml

sample config.yaml, in which user must decide:

• To use GENCODE or RefSeq annotation GTF files.
• Which is the appropriate reference genome for this analysis. Please, read the Heng Li reference and this Biostar answer before taking a final decision.
• Which cutadapt and STAR parameters desire to select
• Which RNA-seq samples user wants to align. In this case we are using E-MTAB-2836.

annotations:
    GRCh38:
        g34:
            url: ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_34/gencode.v34.primary_assembly.annotation.gtf.gz # GENCODE 34 GRCh38 gtf annotation file
            enabled: True # Enabled to be runned
        rs109:
            url: ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.39_GRCh38.p13/GRCh38_major_release_seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_full_analysis_set.refseq_annotation.gtf.gz  
            enabled: True
    GRCh37:
        g19:
            url: XXXX # GENCODE 19 GRCh37 gtf annotation file
            enabled: False
genomes:
    GRCh38:
        url: ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.28_GRCh38.p13/GRCh38_major_release_seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz 
    GRCh37: 
        url: ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/technical/reference/human_g1k_v37.fasta.gz
params:
    cutadapt_pe: "option1"
    STAR: "option2"
samples:
     E-MTAB-XXXX: # proyect
         ERRXXXXX: # sample
             "1": # replicate
                 r1: ERR315325_1.fastq.gz # FastQ read 1
                 r2: ERR315325_2.fastq.gz # FastQ read 2

Finally, execute workflow, deploy software dependencies via conda:

snakemake -n --use-conda

If Slurm has been installed in the computing environment:

snakemake -n --use-conda --profile slurm

Directory structure

├── config
│   └── config.yaml
├── envs
│   └── environment.yaml
├── log
├── out
├── README.md
├── seq
└── Snakefile

Author information and license

Fernando Pozo (@fpozoca – fpozoc@cnio.es) and Tomás Di Domenico.

Project initially forked from here.

Release History

• 1.0.0.

Contributing

1. Fork it (https://gitlab.com/fpozoc/appris_rnaseq.git)
2. Create your feature branch (git checkout -b feature/fooBar)
3. Commit your changes (git commit -am 'Add some fooBar')
4. Push to the branch (git push origin feature/fooBar)
5. Create a new Pull Request