# # MegaLTR

MegaLTR is a robust online server and local standalone that can identifies intact LTR-RTs and calculate LTR Assembly Index (LAI) in any target genome. MegaLTR is freely available at https://bioinformatics.um6p.ma/MegaLTR

MegaLTR is a pipeline that detects intact LTR-RTs at the whole genome level. The pipeline integrates the structure-based, homology-based and de novo intact LTR-RT identification, classification, annotation and visualization tools such as LTR_FINEDR, LTRharvest, LTR_retriever, RepeatMasker, CDHIT package, BLAST+ package, HMMER package, LTRdigest, TEsorter, l REANNOTATE, ClustalW, faidx, Rscript, and RIdeogram.

MegaLTR running with three options:

1: Intact LTR-RT identification and annotation of internal domains.

2: Intact LTR-RT Identification and annotation of internal domains plus determination of insertion time.

3: Intact LTR-RT Identification, annotation of internal domains, determination of insertion time, LTR-RT gene-chimera analysis and visualization of gene density and LTR-RTs across chromosomes.

4: LTR Assembly Index (LAI) calculation.

MegaLTR has been tested on Ubuntu 18.04 and 20.04.

1. Install
2. Required data
3. Usage
4. Run Example
5. Output files
6. Output files example #

   Install

   The installation require conda. You can install all dependencies for running MegaLTR in a new conda environment using the MegaLTR.yml file. If you do not have conda, please follow this tutorial.

1- Download repository from github

git clone https://github.com/MoradMMokhtar/MegaLTR.git

2- Go to the MegaLTR folder

cd MegaLTR

3- Create the MegaLTR environment with all dependencies

conda env create -f MegaLTR.yml

4- Activate the MegaLTR environment

conda activate MegaLTR

Required data

Usage

Go to the MegaLTR folder

bash MegaLTR.sh -A [1 or 2 or 3 or 4] -F [Genome in FASTA Format] -G [GFF/GFF3 File]

#Required arguments:
-A      The analysis type [1 or 2 or 3] 
        1 (for Intact LTR-RT identification and annotation of internal domains 'This analysis needs FASTA file only') 
        2 (for Intact LTR-RT Identification and annotation of internal domains plus determination of insertion time 'This analysis needs FASTA file only') 
        3 (for Intact LTR-RT Identification, annotation of internal domains, determination of insertion time, LTR-RT gene-chimera analysis and visualization of gene density and LTR-RTs across chromosomes 'This analysis needs FASTA and GFF files') 
        4 (for calculate LTR Assembly Index (LAI)'This analysis needs FASTA file only')    
-F      Your path to the genome sequence (Fasta file). 
-G      Your path to the genome annotation (GFF/GFF3 file). Required with argument -A 3 only.

#Optional arguments:
-T      tRNA sequence file (Locate the filename from the tRNA folder or provide your own tRNA sequence in FASTA format, default is Arabidopsis_thaliana_trna.fa)."
-P      Outfileprefix, default is results."
-l      Min length of 5'&3'LTR, default is 100."
-L      Max length of 5'&3'LTR, default is 7000."
-d      Min distance between 5'&3'LTR, default is 1000."
-D      Max distance between 5'&3'LTR, default is 15000."
-S      Specify similaritythreshold, default is 85."
-M      Min length of exact match pair, default is 20."
-B      TE Database that TEsorter will use it {gydb,rexdb,rexdb-plant,rexdb-metazoa,sine}, default is rexdb."
-C      Mininum coverage for protein domains in HMMScan output, default is 20."
-V      Maxinum E-value for protein domains in HMMScan output, default is 0.001."
-Q      Classifying rule [identity-coverage-length] based on similarity, default is 80-80-80]."
-E      Hmm-database that TEsorter will use it {gydb,rexdb,rexdb-plant,rexdb-metazoa,sine}, default is rexdb]."
-R      Neutral mutation rate of the target species (per bp per ya), e.g., rice: 1.3e-8 [0.000000013]; mammal: 2.2e-9 [0.0000000022]; Drosophila: 1.6e-8 [0.000000016], default is 0.000000013."
-U      The distance upstream LTR retrotransposons, default is 5000."
-X      The distance downstream LTR retrotransposons, default is 5000."
-W      Window size to extract gene density from the GFF file, default is 1000000."
-N      Number of chromosomes specified in FASTA file to visualize density of genes and LTRs, default is 12."
-v      Print MegaLTR version and exit."
-t      Indicate how many CPU/threads you want to run MegaLTR, default is 4."
-h      Print this Help

Default parameters:bash MegaLTR.sh -A 3 -F /path/to/genome_fasta_file  -G /path/to/gff_file -T Arabidopsis_thaliana_trna.fa -P Results -l 100 -L 7000 -d 1000 -D 15000 -S 85 -M 20 -B rexdb -C 20 -V 0.001 -Q 80-80-80 -E rexdb -R 0.000000015 -U 500 -X 5000 -W 1000000 -N 9 -t 6

Run Example

bash MegaLTR_Run_Example.sh -A 3 -F NC_003070.9_Arabidopsis_thaliana.fna.gz -G Arabidopsis_thaliana.gff.gz

Output files

We have collected the main output files in the Collected _Files folder in the main output directory. The results are presented in the form of tables and images as follows:

#

Output files example (images)

• Genes and LTR-RTs density across chromosomes

• Boxplot of LTR-RTs length for all LTR-RTs

• Statistical distribution of LTR-RTs length for all LTR-RTs
• Boxplot of LTR-RTs insertion age for all LTR-RTs
• Statistical distribution of LTR-RTs insertion age for all LTR-RTs
• Boxplot of LTR-RT length for each LTR-RTs class
• Statistical distribution of LTR-RT length for each LTR-RTs class
• Boxplot of LTR-RTs insertion age for each LTR-RTs class
• Statistical distribution of LTR-RTs insertion age for each LTR-RTs class #

  For more information and help

To report bugs and give us suggestions, you can open an issue here. You may also contact us by e-mail morad.mokhtar@um6p.ma or achraf.elallali@um6p.ma

If you used MegaLTR to calculate LTR Assembly Index (LAI) please cite PlantLAI (https://doi.org/10.1093/aobpla/plad015) and MegaLTR (https://doi.org/10.3389/fpls.2023.1237426) otherwise please cite MegaLTR (https://doi.org/10.3389/fpls.2023.1237426) and PltRNAdb (https://doi.org/10.1371/journal.pone.0268904)