RiboseqTools

Tools for ribosome profiling data analysis and plot.

Prerequisite

• bowti2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)

• sambamba (http://lomereiter.github.io/sambamba/)

• perl modules

  • BioPerl
  • Getopt::Long
  • Statistics::Basic
  • Statistics::R
  • Term::ANSIColor
  • Bio::Tools::CodonTable

• Ribodiff (https://github.com/ratschlab/RiboDiff) or Riborex (https://github.com/smithlabcode/riborex)

• DESeq2

• R libraries

  • ggplot2
  • reshape
  • gridExtra

Useage:

 perl Riboseq_tools.pl  [task]  [options]

Task:

• map
• meta
• plot
• diff

options:

Global parameters:

-dir      output dir, default: RF_output
-prefix   prefix of output, default: riboseq.
-threads  threads number, default: 10.

** map **

This command creates a modified cds or intron-free genome for reads mapping, but only cds sequences are used for downstream analyses.

-read	  reads file,compressed or not,separated by comma, eg: read1.fq,read2,fq [required]   
-genome   genome file in fasta format [required]
-gff      gff file [required]
-type     mappting to cds or intron-free genome, options: cds,genome. Default: cds
-ext      N bp upstream and downstream sequences to be added to CDS, default: 45	
-mapq     minimal mapping quality, default: 20
-mis      maximal mismatch allowed, default: 1
-seed     seed length for bowtie2, default: 22

** meta **

This command evaluates the patterns of data, eg: phasing, reads coverage and codon enrichment. Figures are generated automatically.

-bam      sorted bam files, separated by comma, eg: bam1,bam2 [required]
-cds      cds sequence file generated by 'map' command [required]
-rdRange  read length ranges for codon analysis, format: num-num. Default: 27-30
-cdRange  codon range to count, cannot > ("ext"/3), format: neg,pos. Default: -15,15
-minCount minimal count of a gene to use, default: 20

** plot **

Plot data generated by meta command, one bam file one graph (.pdf)

-bam      bam files used in meta command, separated by comma, eg: bam1,bam2 [required]

** diff **

This comman calculates differential expression/translation using DESeq2 (for mRNA/RFP) or Ribodiff (for TE). TE can also be calculated using Riborex, which is faster but cannot be applied when the number of samples are different for mRNA and RFP.

    -cds      cds sequence file generated by 'map' command [required]
    -rfLen    reads within this range to count for Riboseq, default: 27-30
    -rsLen    reads within this range to count for mRNAseq, default: 27-30
    -strand   mappng strand to count: +: 0; -: 1; both: 2. Default: 2
    -engine   tool to be used for differential TE analysis, ribodiff or riborex. Default: ribodiff
    -bamRFCnt bam files of riboseq used as control, separated by comma. [required]
    -bamRFTrt bam files of riboseq used as treatmeat. [required]
    -bamRSCnt bam files of mRNAseq used as control. [required]
    -bamRSTrt bam files of mRNAseq used as treatmeat. [required]
    -rdmin    minimal sum of normalized reads for the test. default: 10

options for ribodiff

    -src      location for ribodiff script (TE.py), defualt: utils/TE.py
    -disp     dispersion for riboseq or mRNAseq. 1: different dispersion; 0: same dispersion. default
    -padj     method for multiple test correction, BH, Bonferroni. Default: BH
    -plot     make plots to show the data and results. On: 1; Off: 0. Defulat: 0
    -minP     FDR cutoff for plot. Default: 0.1

options for riborex

    -tool     tools for estimation, options: DESeq2, edgeR, edgeRD, Voom. Default: DESeq2.