RNAcompete

Data analysis scripts for RNAcompete.

Input file

• The input file of the analysis is named raw_data.txt. The first column of raw_data.txt indicates the probe ID, and microarray data for each RBP is listed in two columns: one consists of quantified fluorescence signals captured in TIFF images, and the "FLAG" column indicates "poor quality" spots in microarray.

• We included an example raw_data.txt file in folder /RNAcompete/Normalization/run_normalization.

Analyzing RNAcompete data

1. Download

Clone or download the code and move the /RNAcompete folder to $HOME directory.

2. Install dependencies

• Perl 5

  Add our perl scripts to $PATH and Perl library:

  export PATH=$HOME/RNAcompete/perl5/perl_tools:$PATH;
  export PERL5LIB=$HOME/RNAcompete/perl5/perl_tools:$HOME/RNAcompete/perl5/modules:$PERL5LIB
  

• Matlab 2015b

• R

• ImageMagick

• REDUCE Suite 2

  • Download the version compatible with your system and move the /REDUCE_Suite folder to ~/RNAcompete.

  • Check README by running:

    cd ~/RNAcompete/REDUCE_Suite/bin; ./REDUCE_Suite_setup
    

  • Run the following command to set up REDUCE Suite:

    export REDUCE_SUITE=$HOME/RNAcompete/REDUCE_Suite;
    export PATH=$HOME/RNAcompete/REDUCE_Suite/bin:$PATH
    

  • [optional] The default colors for nucleotides are A, green; C, blue; G, orange; U, cyan. Users may customize the coloring scheme by editing the following code in ~/RNAcompete/REDUCE_Suite/html/LogoGenerator_PS.def:

    /colorDict <<
    (A) green       (a) m_green
    (C) blue        (c) m_blue
    (G) orange      (g) m_orange
    (T) red         (t) m_red
    (U) cyan        (u) m_cyan
    (X) white
    >> def
    

3. Normalization

• (The raw data file we use is the example raw_data.txt file in ~/RNAcompete/Normalization/run_normalization/)

• Copy and paste everything in ~/RNAcompete/Normalization/normalization_scripts/ to ~/RNAcompete/Normalization/run_normalization/ by running:

  cp ~/RNAcompete/Normalization/normalization_scripts/* ~/RNAcompete/Normalization/run_normalization/
  

• Run normalization from /run_normalization directory:

  cd ~/RNAcompete/Normalization/run_normalization/;
  ./scripts.sh
  

4. Motif Generation

RBP information setup

• Move the normalized intensity file (PhaseVII_mad_col_quant_trim_5.txt) to motif generation directories:

  mkdir ~/RNAcompete/RNAcompete_motifs/Data/normalized_probe_scores;
  mv ~/RNAcompete/Normalization/run_normalization/PhaseVII_mad_col_quant_trim_5.txt ~/RNAcompete/RNAcompete_motifs/Data/normalized_probe_scores/
  

• Add information of RBP of interest to info.tab and info_all.tab files in ~/RNAcompete/RNAcompete_motifs/Data

• Edit ~/RNAcompete/RNAcompete_motifs/Data/id.lst to include only RBPs of interest (one RBP_id per line)

Calculate 7mer scores

cd ~/RNAcompete/RNAcompete_motifs/Data/Training_Data/; make maker; make doit

Calculate motifs

cd ~/RNAcompete/RNAcompete_motifs/Predictions/pwm_topX_w7/; make maker; make doit

Create motif logos

cd ~/RNAcompete/RNAcompete_motifs/Figures/Logos/; make maker; make doit

Generate IUPAC motifs

cd ~/RNAcompete/RNAcompete_motifs/IUPACs/pwm_topX_w7/; make maker; make doit

Create HTML output

cd ~/RNAcompete/RNAcompete_motifs/HTML_Reports/; make maker; make doit; make RNAcompete_report_index.html

Related Publications

• D. Ray, K.C. Ha, K. Nie, H. Zheng, T.R. Hughes, Q.D. Morris, RNAcompete methodology and application to determine sequence preferences of unconventional RNA-binding proteins, Methods 118 (2017), 3-15. [Pubmed]

• D. Ray, H. Kazan, K.B. Cook, M.T. Weirauch, H.S. Najafabadi, X. Li, S. Gueroussov, M. Albu, H. Zheng, A. Yang, H. Na, M. Irimia, L.H. Matzat, R.K. Dale, S.A. Smith, C.A. Yarosh, S.M. Kelly, B. Nabet, D. Mecenas, W. Li, R.S. Laishram, M. Qiao, H.D. Lipshitz, F. Piano, A.H. Corbett, R.P. Carstens, B.J. Frey, R.A. Anderson, K.W. Lynch, L.O. Penalva, E.P. Lei, A.G. Fraser, B.J. Blencowe, Q.D. Morris, T.R. Hughes, A compendium of RNA-binding motifs for decodng gene regulation, Nature 499(7457) (2013) 172-7. [Pubmed]

• D. Ray, H. Kazan, E.T. Chan, L. Pena Castillo, S. Chaudhry, S. Talukder, B.J. Blencowe, Q. Morris, T.R. Hughes, Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins, Nature biotechnology 27(7) (2009) 667-70. [Pubmed]

For questions, please email kun.nie[at]mail.utoronto.ca