NLR-Annotator
Introduction
NLR-Annotator is a tool to annotate loci associated with NLRs in large sequences. It is searching for amino acid motifs within all 6 frames of a nucleotide sequence. An NLR locus is defined from first to last motif that can be associated with an NLR. It does NOT predict genes. A predicted NLR locus might be a pseudogene. if it is overlapping with a gene, the actual gene start or intron-exon boundaries are not given. It just points you to the loci that might be worth investigating, which we hope you will find useful. Details are published in Steuernagel et al.: The NLR-Annotator tool enables annotation of the intracellular immune receptor repertoire, Plant Physiology, 2020
Workflow
The NLR-Annotator pipeline consists of three steps:
- Chopping the input sequence into overlapping sub-sequences. This allows the usage of the Motif Alignment Search Tool (MAST) and also allows parallelization. The sub-sequences are overlapping to ensure no locus is missed because it was split by the chopping.
- Running NLR-Parser. The chopped sub sequences are searched for NLR-associated motifs. The motifs are defined by Jupe et al. 2012 (Table 1). The concept of NLR-Parser has been published but for NLR-Annotator, the updated version bundled with this software is required.
- Running NLR-Annotator. This program integrates the motifs annotated in the sub-sequences and searches for the actual NLR-Loci. Several output formats are provided.
Requirements
MEME suite
The MEME suite is available at http://meme-suite.org/index.html
Don't worry about setting up the Apache webserver. You just need MAST, so the quick install is sufficient.
NLR-Parser in the current release is not compatible with meme-suite versions above 4.9.1. If you want to use a newer version of meme-suite, please switch to the branch nlr_parser3.
JRE 1.6
Make sure you have the Java Runtime Environments 1.6 or higher. Download from http://java.com
Installation
Make sure MEME suite and JRE are installed. Download three jar files (ChopSequence.jar, NLR-Parser.jar and NLR-Annotator.jar) as well as the motif definition file (meme.xml) from the release. Done.
Running NLR-Annotator pipeline
Chopping sequences.
Input sequences are required to be nucleotide sequences and in FASTA format. Files may be gzip compressed.
Usage
java -jar ChopSequence.jar -i <inputsequence.fasta> -o <outputsequence.fasta> -l <sub-sequence length> -p <length of overlap>
Parameters
| parameter | argument |
|---|---|
| -i | input file with sequence in fasta format |
| -o | output file with sequence in fasta format |
| -l | (integer) length of sub-sequence (default 20000) |
| -p | (integer) length of overlap (default 5000) |
NLR-Parser
Usage
java -jar NLR-Parser.jar -t <number of threads> -y <path/to/meme/bin/mast> -x <path/to/meme.xml> -i <sub-seqeunces.fasta> -c <output.nlr.xml>
| parameter | argument |
|---|---|
| -x | The path to the meme.xml (The motif definitions) |
| -y | The path to the mast installation (including the mast command. e.g. /programs/meme/bin/mast ) |
| -i | The file with the chopped sequences. (Output from ChopSequence.jar) |
| -c | The output file that will be created. This is in xml format and will be the input for NLR-Annotator. |
NLR-Annotator
Usage
java -jar NLR-Annotator.jar -i <nlr.xml> -o <output.nlr.txt>
| parameter | argument | description |
|---|---|---|
| -i | input.xml | Input file in xml format. This is what comes out from NLR-Parser -c of the chopped inputSequence |
| -o | output.txt | output file in tabular format |
| -g | output.gff | output file in gff format |
| -b | output.bed | output file in bed format |
| -m | output.motifs.bed | output file of the motifs in bed format |
| -a | output.nbarkMotifAlignment.fasta | output file of the nb-arc motifs as multiple alignment. This file can be used as input to generate a phylogenetic tree. |
| -f | genome.fasta output.nlr.fasta flanking | Write fasta of nlr loci. This parameter requires 3 arguments. The first is the original (not chopped) input sequence. The second is the file that is being generated. The third is the length of flanking sequence around the loci. |
| -distanceWithinMotifCombination | integer | (default:500) |
| -distanceForElongating | integer | (default:2500) |
| -distanceBetweenMotifCombinations | integer | (default:10000) |