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

morrislab/RNAcompete