GitHub Conventions:
   - Master branch is the current release
   - Development branch is not gauranteed to be stable
   - Source releases are avaialable under the GitHub release tab and at 
     http://www.repeatmasker.org

RepeatModeler

RepeatModeler is a de novo transposable element (TE) family identification and modeling package. At the heart of RepeatModeler are three de-novo repeat finding programs ( RECON, RepeatScout and LtrHarvest/Ltr_retriever ) which employ complementary computational methods for identifying repeat element boundaries and family relationships from sequence data.

RepeatModeler assists in automating the runs of the various algorithms given a genomic database, clustering redundant results, refining and classifying the families and producing a high quality library of TE families suitable for use with RepeatMasker and ultimately for submission to the Dfam database ( http://dfam.org ).

Authors

RepeatModeler: Robert Hubley, Arian Smit - Institute for Systems Biology

LTR Pipeline Extensions: Jullien M. Flynn - Cornell University

Installation

There are two supported paths to installing RepeatModeler on a UNIX-based server. RepeatModeler may be installed from source as described in the “Source Distribution Installation” instructions below, or using one of our Dfam-TETools container images ( Docker or Singularity ). The containers include RepeatModeler, it’s prerequisites and additional TE analysis tools/utilities used by Dfam. Information on the Dfam-TETools container may be found here: https://github.com/Dfam-consortium/TETools

Source Distribution Installation

Prerequisites

Perl Available at http://www.perl.org/get.html. Developed and tested with version 5.8.8.

RepeatMasker & Libraries Developed and tested with 4.1.9. The program is available at http://www.repeatmasker.org/RMDownload.html and is distributed with Dfam - an open database of transposable element families.

RECON - De Novo Repeat Finder, Bao Z. and Eddy S.R. Developed and tested with our patched version of RECON ( 1.08 ). The 1.08 version fixes problems with running RECON on 64 bit machines and supplies a workaround to a division by zero bug along with some buffer overrun fixes. The program is available at: http://www.repeatmasker.org/RECON-1.08.tar.gz. The original version is available at http://eddylab.org/software/recon/.

RepeatScout - De Novo Repeat Finder, Price A.L., Jones N.C. and Pevzner P.A. Developed and tested with our extended version of RepeatScout ( 1.0.7 ). This version is available at https://github.com/Dfam-consortium/RepeatScout

TRF - Tandem Repeat Finder, G. Benson et al. You can obtain a free copy at http://tandem.bu.edu/trf/trf.html RepeatModeler requires version 4.0.9 or higher.

RMBlast - A modified version of NCBI Blast for use with RepeatMasker and RepeatModeler. Precompiled binaries and source can be found at http://www.repeatmasker.org/RMBlast.html We highly recommend using 2.14.1 or higher.

RepeatAfterMe - An automated MSA extension program for TE families. This is available for download at: https://github.com/Dfam-consortium/RepeatAfterMe. We recommend 0.0.6 or higher.

UCSC genome browser command-line utilities - Some tools included with RepeatModeler work with files in the ‘twobit’ file format using these programs: twoBitToFa, faToTwoBit, and twoBitInfo. Precompiled binaries and source for these programs can be found at http://hgdownload.soe.ucsc.edu/downloads.html#utilities_downloads.

Optional. Required for running LTR structural search pipeline:

LtrHarvest - The LtrHarvest program is part of the GenomeTools suite. We have developed this release of RepeatModeler on GenomeTools version 1.5.9 available for download from here: http://genometools.org/pub/ NOTE: use the “make threads=yes” build options to enable multi-threaded runs.

Ltr_retriever - A LTR discovery post-processing and filtering tool. We currently require the 2.9.0 version of LTR retreiver, newer versions of LTR_retriever will not currently work with RepeatModeler. https://github.com/oushujun/LTR_retriever/releases

MAFFT - A multiple sequence alignment program. We developed and tested RepeatModeler using mafft version 7.505. Please use this verison or higher from here: https://mafft.cbrc.jp/alignment/software/

CD-HIT - A sequence clustering package. We developed and tested RepeatModeler using version 4.8.1. Please use this version or higher from: https://github.com/weizhongli/cdhit

Ninja - A tool for large-scale neighbor-joining phylogeny inference and clustering. We developed and tested RepeatModeler using Ninja version “0.98-cluster_only”. Please obtain a copy from: https://github.com/TravisWheelerLab/NINJA/releases/tag/0.98-cluster_only

Installation

Obtain the source distribution
- Github : https://github.com/Dfam-consortium/RepeatModeler Available by cloning the master branch of the RepeatModeler repository ( latest released version ) or by downloading a release from the repository “releases” tab. or
- RepeatMasker Website : http://www.repeatmasker.org/RepeatModeler
Uncompress and expand the distribution archive:
```
tar -zxvf RepeatModeler-#.#.tar.gz
```
Configure for your site:

Automatic:
- Run the “configure” script interactively with prompts for each setting:
```
  perl ./configure
```
- Run the “configure” script with supplied paramters:
```
  perl ./configure -rscout_dir .. -recon_dir ..
```
By Hand:
- Edit the configuration file “RepModelConfig.pm”
Dynamically:
- Use the “configuration overrides” command line options with the RepeatModeler programs. e.g:
```
  ./RepeatModeler -rscout_dir .. -recon_dir .. 
```

Example Run

In this example we first downloaded elephant sequences from Genbank ( approx 11MB ) into a file called elephant.fa.

Create a Database for RepeatModeler

RepeatModeler uses a NCBI BLASTDB as input to the repeat modeling pipeline. A utility is provided to assist the user in creating a single database from several types of input structures.
```
  <RepeatModelerPath>/BuildDatabase -name elephant elephant.fa
```
Run “BuildDatabase” without any options in order to see the full documentation on this utility. There are several options which make it easier to import multiple sequence files into one database.

TIP: It is a good idea to place your datafiles and run this
```
 program suite from a local disk rather than over NFS. 
 This will greatly improve runtime as the filesystem 
 access is considerable
```
Run RepeatModeler

RepeatModeler runs several compute intensive programs on the input sequence. For best results run this on a single machine with a moderate amount of memory > 32GB and multiple processors.
Our setup is Xeon(R) CPU E5-2680 v4 @ 2.40GHz - 28 cores, 128GB RAM. To specify a run using 20 threads (at most), and including the new LTR discovery pipeline:
```
   nohup <RepeatModelerPath>/RepeatModeler -database elephant 
   -threads 20 -LTRStruct >& run.out &
```
The nohup (or screen) is used on our machines when running long jobs. The log output is saved to a file and the process is backgrounded. For typical runtimes ( can be > 1-2 days with this configuration on a well assembled mammalian genome ) see the run statistics section of this file. It is important to save the log output for later usage.
It contains the random number generator seed so that the sampling process may be reproduced if necessary. In addition the log file contains details about the progress of the run for later assesment of peformance or debuging problems.
Interpret the results

RepeatModeler produces a voluminous amount of temporary files stored in a directory created at runtime named like:
```
      RM_<PID>.<DATE> ie. "RM_5098.MonMar141305172005" 
```
and remains after each run for debugging purposes or for the purpose of resuming runs if a failure occures. At the succesful completion of a run, three files are generated:
```
      <database_name>-families.fa  : Consensus sequences
      <database_name>-families.stk : Seed alignments
      <database_name>-rmod.log     : A summarized log of the run
```
The seed alignment file is in a Dfam compatible Stockholm format and may be uploaded to the Dfam database by submiting the data to help@dfam.org or by going to dfam.org/login and creating an upload account.

The fasta format is useful for running quick custom library searches using RepeatMasker. Ie.:
```
      <RepeatMaskerPath>/RepeatMasker -lib <database_name>-families.fa mySequence.fa 
```
Other files produced in the working directory include:

       RM_<PID>.<DATE>/
          consensi.fa
          families.stk
          round-1/
               sampleDB-#.fa       : The genomic sample used in this round
               sampleDB-#.fa.lfreq : The RepeatScout lmer table
               sampleDB-#.fa.rscons: The RepeatScout generated consensi
               sampleDB-#.fa.rscons.filtered : The simple repeat/low 
                                               complexity filtered
                                               version of *.rscons
               consensi.fa         : The final consensi db for this round
               family-#-cons.html  : A visualization of the model
                                     refinement process.  This can be opened
                                     in web browsers that support zooming.
                                     ( such as firefox ).
                                     This is used to track down problems 
                                     with the Refiner.pl
               index.html          : A HTML index to all the family-#-cons.html
                                     files.
          round-2/
               sampleDB-#.fa       : The genomic sample used in this round
               msps.out            : The output of the sample all-vs-all 
                                     comparison
               summary/            : The RECON output directory
                    eles           : The RECON family output
               consensi.fa         : Same as above
               family-#-cons.html  : Same as above
               index.html          : Same as above
          round-3/
               Same as round-2
           ..
          round-n/

Recover from a failure

If for some reason RepeatModeler fails, you may restart an analysis starting from the last round it was working on. The -recoverDir [ResultDir] option allows you to specify a diretory ( i.e RM_./ ) where a previous run of RepeatModeler was working and it will automatically determine how to continue the analysis.

Caveats

RMBlast uses the NCBI stat reporting mechanism to report usage statistics back over the net. If RepeatModeler is taking in inordinate amount of time to complete ( > 1 week on a multi-core machine ) or you do not have an outside network connection to the machine, you should disable this reporting feature by setting the environment variable BLAST_USAGE_REPORT=false or by creating a .ncbirc file in the users home directory with the stanza:

[BLAST]

BLAST_USAGE_REPORT=false
RepeatModeler is designed to run on assemblies rather than genome reads. At the start of a run a quick analysis is performed on the input database to ascertain the assembly N50. A histogram of contig size is also displayed.
RepeatModeler employs symmetric multiprocessing parallelism, therefore it should be run on a single machine per-assembly.
It is not recommended that a genome be run in a batched fashion nor the results of multiple RepeatModeler runs on the same genome be naively combined. Doing so will generate a combined library that is largely redundant. The -genomeSampleSizeMax parameter is provided for the purpose of increasing the amount of the genome sampled while avoiding rediscovery of families.

Please see the RELEASE-NOTES file for more details.

RepeatModeler Statistics

Benchmarks and statistics for runs of RepeatModeler on reference genomes.

RepeatModeler 2.0.7 ( RECON + RepeatScout + LTRStruct ) 48 threads:

Genome	Genome DB Size (bp)	Runtime (hh:mm)*	Models Built
D. melanogaster	164 Mbp	3:41	492
H. sapiens	3.1 Gbp	21:26	810

Analysis run on a Ubuntu 22.04.3 LTS Linux system with Intel(R) Xeon(R) CPU E5-2680 v4 @ 2.40GHz processors. Both genomes were run with “-threads 48”.



Credits
-------

Arnie Kas for the work done on the original MultAln.pm.

Andy Siegel for statistics consultations.

Thanks so much to Warren Gish for his invaluable assistance 
and consultation on his ABBlast program suite.

Alkes Price and Pavel Pevzner for assistance with RepeatScout
and hosting my multi-sequence version of RepeatScout.

Shujun Ou, and Ning Jiang for discussions and assistance with 
using LTR_retreiver.

This work was supported by the NIH ( R44 HG02244-02), 
( RO1 HG002939 ), ( U24 HG010136 ), and the Institute 
for Systems Biology.


License
-------

This work is licensed under the Open Source License v2.1.  
To view a copy of this license, visit 
http://www.opensource.org/licenses/osl-2.1.php or
see the LICENSE file contained in this distribution.