Commit 2c162ba7 authored by harm's avatar harm
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### README #####################################################################
This R-script software serves to predict guides for RfxCas13d Type-IV CRISPR proteins to maximaize knock-down efficiencies.
Guide efficacies are modeled after an on-target tilling screen against GFP CD46 CD55 and CD71 mRNA.
If you are using our software, please cite:
Hans-Hermann Wessels, Alejandro Méndez-Mancilla, Xinyi Guo, Mateusz Legut, Zharko Daniloski, Neville E. Sanjana.
Principles for rational Cas13d guide design. bioRxiv (2019). doi:10.1101/2019.12.27.889089
This R-script software serves to predict guides for RfxCas13d Type-IV CRISPR proteins to maximize knock-down efficiencies.
Guide efficacies are modeled after an on-target tilling screen against GFP, CD46, CD55, and CD71 mRNA.
# Installation
Please, first install RNAhybrid.
You have already unpacked Cas13design.tar.gz
tar -xzf Cas13design.tar.gz
Unpacking Cas13design.tar.gz will create a Cas13design directory, as well as scripts and data sub-directories.
The main executable RfxCas13d_GuideScoring.R can be found within the scripts sub-directory.
External RNAfold and RNAplfold binaries v2.4.10 are included. RNAhybrid may be installed and added to your $PATH (see below).
All R packages needed will be automatically installed within the scripts folder upon the first execution.
This may increase the runtime of the first run by several minutes. We recommend using R v3.6 or newer to execute RfxCas13d_GuideScoring.R.
If older R versions are used, the Bioconductor package Biostrings may need to be installed manually via BiocLite.
Model input data as well as an example input fasta file are included within the data sub-sirectory.
Next, please install RNAhybrid.
The source code can be downloaded here:
"https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid?id=rnahybrid_view_download"
wget https://bibiserv.cebitec.uni-bielefeld.de/applications/rnahybrid/resources/downloads/RNAhybrid-2.1.2.tar.gz
copy RNAhybrid-2.1.2.tar.gz into ./scripts/ and unpack RNAhybrid
copy RNAhybrid-2.1.2.tar.gz into ./Cas13design/scripts/ and unpack RNAhybrid
cp RNAhybrid-2.1.2.tar.gz ./scripts/
cd ./scripts/
cp RNAhybrid-2.1.2.tar.gz ./Cas13design/scripts/
cd ./Cas13design/scripts/
tar -xzf RNAhybrid-2.1.2.tar.gz
cd RNAhybrid-2.1.2
Follow the installation instructions for RNAhybrid in INSTALL:
If you install it on your system and you have sudo rights, do:
If you install it on your local system and you have sudo rights, do:
./configure
make
make install
......@@ -33,7 +57,7 @@ Follow the installation instructions for RNAhybrid in INSTALL:
./configure --prefix=$HOME #this will create a bin folder in your $HOME directory
make
make install
export PATH=$HOME/bin:$PATH #You may want to at this to your ~/.bashrc or ~/.bash_profile
export PATH=$HOME/bin:$PATH # You may want to add this to your ~/.bashrc or ~/.bash_profile
Make sure RNAhybrid has been added to your PATH
......@@ -44,23 +68,8 @@ The Software will assume RNAhybrid is in ~/bin/
All other dependencies are included in the Cas13design.tar.gz archive
to unpack run
tar -xzf Cas13design.tar.gz
Unpacking Cas13design.tar.gz will create a Cas13design directory, as well as scripts and data sub-directories.
The main executable RfxCas13d_GuideScoring.R can be found within the scripts sub-directory.
External RNAfold and RNAplfold binaries v2.4.10 are included.
All R packages needed wil be automatically installed within the scripts folder upon te first execution. This may increase the runtime of the first execution by several minutes.
Model input data as well as an example input fasta file are included within the data sub-sirectory.
......@@ -70,7 +79,7 @@ Model input data as well as an example input fasta file are included within the
# Predict guides
To predcit guides simply call RfxCas13d_GuideScoring.R with its three required input arguments in order.
To predict guide RNA efficacies simply call RfxCas13d_GuideScoring.R with its three required input arguments in order. In its current implmentation, the script will run successfully only within its main directory Cas13design.
cd Cas13design
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If you run the script the first time, R package installation may take several minutes.
Once Cas13design is fully installed, the run time scales with the fasta input length.
The minimum length supplied must be at least 100 nucleotides, as the model requires the upstream sequences to compute the upstream U-context.
The limiting step is the crRNA-folding prediction.
The total run time for the provided ~1000nt test.fa example is about 2 min and 10 sec.
The minimum length supplied must be at least 30 nucleotides. The total run time for the provided ~1000nt test.fa example is about 2 min and 10 sec.
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rank
quartile according to input screens
2) A csv file containing all predicted guides
3) if plot was set true, a pdf file is returned depicting the score destribution along the target transcripts
3) if plot was set to TRUE, a pdf file is returned depicting the score destribution along the target transcripts
According to our corresponding study, high confidence guide predictions have standardized guide scores between 0.69 and 0.79 (95% confidence interval).
Guide scores range between a 0 - 1 interval, with higher scores being indicative for higher predicted knock-down efficacy.
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