d3b-center/diseasexpress-pipeline

pipeline to process diseaseXpress datasets

DiseaseXpress RNA-seq Pipeline

Authors:	Komal Rathi
Contact:	[email protected]
Organization:	DBHi, CHOP
Status:	This is "work in progress"
Date:	2024-09-28

Login

ssh -i "rnaseq.pem" [email protected]

Installation

Install all software using conda. Depending on your system, you might have to install other pre-requisites.

conda create --name rnaseq-env
source activate rnaseq-env
conda install -c biobuilds sra-tools=2.5.6
conda install -c bioconda rsem=1.2.28
conda install -c bioconda star=2.5.2b

# R packages to install
GEOquery
SRAdb
DBI

# Other tools required for faster downloads
EDirect: https://www.ncbi.nlm.nih.gov/books/NBK179288/
aspera ascp client: http://downloads.asperasoft.com/en/downloads/50

Pipeline

Create Genome Index for STAR and RSEM:

# This is to be done just once per genome build (hg19, hg38, mm10 etc).
# you need to have an existing fasta and gtf (check config.yaml)
snakemake -p -s Snakefile_genome --config freeze=hg19

Get raw fastq files and create a directory structure:

# provide either GEO accession or SRA study ID
Rscript getSRA.R SRP033200
Rscript getSRA.R GSE52564

This will create a directory structure under the source dir (by default: /mnt/rnaseq/data/raw) like this:

.
├── GSE52564
│   ├── sra
├── SRP033200
│   └── sra
└── log.txt

Run snakemake

Then run snakemake with three parameters:

1. -f or --freeze. The genome build (e.g. mm10, hg19 or hg38).
2. -s or --sourcedir. The source directory which is path to the project directory.
3. -p or --paired. TRUE or FALSE for paired or single-ended reads.

# E.g. to process data in /mnt/rnaseq/data/raw/GSE57945
# for single-ended reads
source activate rnaseq-env
bash run_snakemake.sh -f=hg38 -s=/mnt/rnaseq/data/raw/GSE57945 -p=FALSE

# for paired-end data
bash run_snakemake.sh -f=hg38 -s=/mnt/rnaseq/data/raw/GSE52564 -p=TRUE

This will create an output directory structure like this:

# output directory structure for GSE2564:

tree -L /mnt/rnaseq/data/raw/GSE52564/

├── bam
│   ├── SRR1033783_Aligned.toTranscriptome.out.bam
│   ├── SRR1033783_Log.final.out
│   ├── SRR1033783_Log.out
│   ├── SRR1033783_Log.progress.out
│   ├── SRR1033783_SJ.out.tab
├── fastq
│   ├── SRR1033783_1.fastq.gz
│   ├── SRR1033783_2.fastq.gz
├── quant
│   ├── SRR1033783.genes.results
│   ├── SRR1033783.isoforms.results
│   ├── SRR1033783.stat
│   │   ├── SRR1033783.cnt
│   │   ├── SRR1033783.model
│   │   └── SRR1033783.theta
└── sra
    |── SRR1033783.sra