RiboKit : REEFFIT

RNA Ensemble Extraction From Footprinting Insights Technique

Running REEFFIT

Almost all of REEFFIT's functionality is accessed through the reeffit command, located in the REEFFIT_HOME/bin directory that was added to the PATH variable during installation.

There are four steps that are usually performed in a REEFFIT analysis, all of which are accessed through the reeffit command:

  • Obtaining a secondary structure ensemble
  • Preparing cross-validation or bootstrapping shell files
  • Executing the shell files
  • Compiling the results.

The following subsections explain how to run each.


General Form

In each of the following analyses, the reeffit command is used in various ways. REEFFIT's main input is an RDAT file containing the data and details of the chemical mapping experiment. Besides this, the only other necessary option is the output directory. The general form of the reeffit command is:

reeffit RDAT_FILE OUTPUT_DIR [OPTIONS]

The options given to REEFFIT will dictate its behavior.


Obtaining Structural Ensemble

If a hypothesized secondary structure ensemble is not available, REEFFIT can estimate it using one of several methods:

  • Sampling of the suboptimal ensemble for all sequences (option --modelselect=sample):

Up to 200 suboptimal structures that lie in at most 5% distance to the minimum free energy secondary structure are sampled for each sequence in the ensemble. The full resulting set of structures (usually on the order of 200 to 1000 for RNAs of 100 to 200 nucleotides in length), located in OUTPUT_DIR/all_structures.txt are used for subsequent analyses.

  • Selection of a small number of structures in the suboptimal ensemble that best agree with the data a priori (option modelselect=heuristic):

The same procedure as in the point above is used to obtain a large number of suboptimal structures. These are then clustered into a small number of clusters (calculated by maximizing the Calinksi-Barabasz index) and representatives for each cluster are chosen by scoring them against each chemical mapping profile in the data using 1-dimensional mapping secondary struture directed modeling. Additional high-scoring structures are added if they decrease the AIC information score after a few EM iterations.

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The recomended method to obtain the structural ensemble is the first one: using all the structures sampled. We have observed that this captures most of the data better and can be properly regularized with several priors to account for the large number of variables to fit (see below). Therefore, to obtain the structural ensemble, the reeffit command would be:

reeffit RDAT_FILE OUTPUT_DIR --modelselect=sample

Ensemble Fitting Options

There are several options that affect the way REEFFIT performs the fit to the data. The most important ones to consider are the ones below:

  • Number of parallel jobs (option --njobs):

Some of REEFFIT's calculations can be performed quickly in parallelized form. This option can specify the number of jobs to split the calculations into.

  • Number of EM iterations (option --refineiter):

Number of maximum EM iterations to perform. The default is 10.

  • Mode of inference for the E-step (option --softem to use soft EM (MCMC inference) instead of the default hard EM (MAP estimation) mode):

The most rigorous way to run REEFFIT is using the --softem option to perform MCMC inference at each E-step in the calculation (set the number of simulation steps to take with the --nsim option, which defaults to 1000). This however, is terribly costly computationally and can come at an expense of other important downstream analyses, like bootstrapping. In our experience, performing hard EM does not alter the results terribly and can therefore be safely used.

  • Motif decomposition (option --decompose, default is no decomposition):

When the ensemble to fit is large, it may be useful to decompose the structures into overlapping secondary structure motifs, forcing all motifs that are structurally the same to have the same reactivity profile. This greatly reduces the number of variables in the model and speeds up the computation. Note that this is not activated by default.

  • Data normalization (option --boxnormalize):

When handling capillary electrophoresis chemical mapping data that has not been rigorously normalize (through, for example, a dilution series), it is recommended to normalize the data to conform to the prior reactivities coded into REEFFIT. This option should box the data into values from 0 to 2 and get rid of outliers. Note that if normalization has been previously peformed on the dataset, this additional normalization will produce large artifacts and will essentially "flatten" the data.


Preparing Bootstrap

In order to robustly estimate population fraction errors, we perform boostrapping. Because bootstrapping is computationally expensive, we recommend using the reeffit command to prepare shell "worker" scripts that will perform the bootstrapping in parallel.

To achieve this, we use the --preparebootstrap option. To set up the scripts, we have to divide the number of bootstraps into the number of worker scripts that are going to be running simultaneously using the --nworkers and --ntasks options. For example, to set up 100 bootstraps for 5 workers, we would use the options --nworkers=5 --ntasks=20.

It is important to note that every option passed to the command will be passed to the worker scripts.

Assuming that we have the structural ensemble in OUTPUT_DIR/all_structures.txt and we want to activate motif decomposition, the REEFFIT command for this task would be:

reeffit RDAT_FILE OUTPUT_DIR --structfile=OUTPUT_DIR/all_structures.txt --decompose --preparebootstrap --nworkers=5 --ntasks=20

This will write several bootstrap_workerN.sh scripts to the output directory, as well as a master_bootstrap_script.sh


Executing Bootstrap and Compiling Results

Once the bootstrapping files are set up, we can execute them and compile the results using the master script.

To execute the bootstrap workers prepared in the section above, execute the generated master script:

sh OUTPUT_DIR/master_bootstrap_script.sh execute

All workers will then execute in parallel and store their results in OUTPUT_DIR/bootN directories.

After the workers are done, you can compile their results using the master script as well:

sh OUTPUT_DIR/master_bootstrap_script.sh compile

This will take some time, since it will do a REEFFIT fit will the full data in addition to compile the bootstrapping results.


Generating PDF Report

Optionally, REEFFIT can produce a PDF report of the bootstrap results. This is achieved with the reeffit_report command, which is added to your PATH variable during installation:

reeffit_report OUTPUT_DIR NAME PREFIX

Here, the NAME option is just to give a name to output structures in the report. The PREFIX option specifies which result files REEFFIT will use to generate the report. For example, all result files in the bootstrap analysis by default start with the bootstrap prefix. Therefore, to generate a report using the bootstrap results, PREFIX would be set to bootstrap.

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© Copyright 2013-2017 Pablo Cordero G., Leland Stanford Junior University. All Rights Reserved.

Last updated on Jun 22, 2017.