##R.A.G.E. : Ribosome Assisted Genome Exploration
###Introduction
With the development of high-throughput sequencing technologies in past years, ribosome profiling has emerged as a novel technique that provides a quantitative and qualitative measure of mRNA translation and protein production. Ribosome profiling (or Ribo-Seq) is performed via nuclease footprinting, which involves digestion of all RNA segments unoccupied by the ribosomes. The ribosome protected fragments or “footprints” are purified and then subjected to deep sequencing. Given that this technique is rather new, few genome annotation pipelines have incorporated the information that Ribo-Seq provides into their predictions. RAGE is an automated software pipeline targeted towards prokyaryotic genomes that uses mRNA and Ribo-seq data along with a reference genome to produce a set of gene predictions that are ranked according to which data set aided the gene discovery. This pipeline was original created by the contributors for the course 03-713 at Carnegie Mellon University.
###Requirements
- Linux Operating System (64-bit)
- Python version 2.7 or higher
- BioPython module
- Cutadapt-1.4.2 or higher (for removing the Adaptors and cleaning the reads)
- Bowtie-1.0.1 or higher - (for aligning the reads to the genome)
- Samtools-0.1.19 or higher - (for converting sam files to bam files)
- CEM -v0.9.1 or higher - (for assembling transcripts from RNA-Seq reads)
- glimmer3.02 - (for de novo gene prediction)
- cufflinks-2.2.0 - (for converting gtf to gff3 file)
###Setup
If you haven’t already setup the paths for the tools, run the command below to input the paths directly into the bash_profile file.
python pathhelp.py
###Example Commands
nohup python rage-v2.4.py $HOME/data/ecolik12/k12rnaseq.fastq $HOME/data/ecolik12/k12riboseq.fastq $HOME/data/ecolik12/k12genome.fasta &
nohup python rage-v2.4.py $HOME/data/ecolik12/k12rnaseq.fastq $HOME/data/ecolik12/k12riboseq.fastq $HOME/data/ecolik12/k12genome.fasta -d $HOME/results/data/outputsfinaltest/ -w 9#3#1 -s ecolik12 &
nohup python rage-v2.4.py $HOMEresults/2014-04-07/rnaseqtrimmed.fastq $HOME/results/2014-04-16/outputs/cutadapt/k12riboseq-ribotrimmed.fastq $HOME/data/ecolik12/k12genome.fasta -c false &
nohup python rage-v2.4.py $HOMEresults/2014-04-07/rnaseqtrimmed.fastq $HOME/results/data/outputsfull/filter/ribotrimmedfiltered_rRNA_tRNA.fastq $HOME/data/ecolik12/k12genome.fasta -c false -f false &
nohup python rage-v2.4.py $HOME/results/data/outputfullwglimmer/bowtie/rnaseqtrimmed.sorted.bam $HOME/results/data/outputsfull/rnaseqtrimmed.sam $HOME/data/ecolik12/k12genome.fasta -c false -f false -b false &
###Parameters
Usage: rage-v2.4.py [-h] [-c {true,false}] [-f {true,false}] [-b {true,false}]
[-d WORKDIRCTORY] [-a RNA_ADAPTER] [-i RIBOSOME_ADAPTER]
[-w WEIGHTS] [-s SPECIES]
Positional Arguments:
rnainput input RNA-seq fastq file
riboinput input Ribosome fastq file
genomeinput input Genome fasta file
Optional Arguments:
-h, --help show this help message and exit
-c {true,false}, --cutadapt {true,false}
whether use cutadapt, if choose false, please input
the clean RNA-seq and ribosome fastq file; default is
true
-f {true,false}, --filter_rRNA_tRNA {true,false}
whether filter rRNA and tRNA, if choose false, please
input the clean ribosome fastq file; default is true
-b {true,false}, --bowtie {true,false}
whether use bowtie to map, if choose false, please
input sam file for Ribo-footprinting and sorted .bam
for RNA-seq; default is true
-d WORKDIRCTORY, --WorkDirctory WORKDIRCTORY
rnainput riboinput genomeinput
-a RNA_ADAPTER, --rna_adapter RNA_ADAPTER
input custom adapt sequence for RNA-seq
-i RIBOSOME_ADAPTER, --ribosome_adapter RIBOSOME_ADAPTER
input custom adapt sequence for Ribosome profiling
-w WEIGHTS, --weights WEIGHTS
Assign points for ribo-footprinting, RNAtranscripts
and naiveORFs; format XX#XX#XX
-s SPECIES, --species SPECIES
Species name of the data
###References
####LICENSE This program is free software and is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.
####CONTRIBUTORS
- Dr. C. Joel McManus
- Minh Le
- Easwaran Ramamurthy
- Xinyu Miao
- Wenzhu Liu
- Siddharth Gurdasani
- Patrick Ropp