Package: ncOrtho
ncOrthoNcOrtho is a tool for the targeted search of orthologous micro RNAs (miRNAs) throughout the tree of life.
Conceptually, it works similar to the programfDOGin that a probabilistic model of
a reference miRNA is created. For training the model, orthologs of the reference sequence are first identified in
a set of taxa that are more closely related to the reference species. In contrast to fDOG, ncOrtho does not train hidden
Markov Models but covariance models (CMs) (Eddy & Durbin, 1994)
which also model conservation of the miRNA's secondary structure.How to citeLangschied F, Leisegang MS, Brandes RP, Ebersberger I.ncOrtho: efficient and reliable identification of miRNA orthologs.
Nucleic Acids Res. 2023 Jun 1:gkad467.
doi: 10.1093/nar/gkad467.
Epub ahead of print. PMID: 37260093.Getting StartedNcOrtho depends on multiple third party applications, some of which are Linux specific.
All dependencies can be installed withAnaconda.
It is recommended to create a new Anaconda environment for this. For example:conda create --name ncOrtho python=3.8
conda activate ncOrthoPrerequisitesOperating System:Linux (tested on: Ubuntu 20.04)Python:version 3 or higher (tested with v3.8)ToolTested versionAnaconda installationBLASTnv2.7.1conda install -c kantorlab blastnInfernalv1.1.4conda install -c bioconda infernalt_coffeev13.45conda install -c bioconda t-coffeeMUSCLEv5.1conda install -c bioconda muscleInstallingAfter installing all three dependencies, ncOrtho can be installed withpip:pip install ncOrthoUsageCM constructionAs a targeted search for orthologs, ncOrtho's biggest strength is its flexibility to change the taxonomic
scope of an analysis according to the research question at hand.For this reason, a few questions need to be answered, before we can start constructing covariance models:What is the reference species?How phylogenetically diverse will my set of target species be?From which species should the core set of miRNA orthologs be extracted, which will be used for training the CMs?Which miRNAs are going to be used for the ortholog search?To identify suitable core species for a given reference species you can calculate an estimate of conserved synteny
given a set of pairwise ortholog predictions with:ncCheck -p <parameters.yaml> -o <outdir>You can find additional information about ncCheck in theWIKI.As soon as you know what your core species are going to be, you will need to collect the following data:Genomic sequence in FASTA format (e.g "genomic.fna" from RefSeq)Genome annotation in GFF3 format (e.g. "genomic.gff" from RefSeq)Pairwise orthologs of all proteins between the reference and each core species (more informationhereModify theexample parametersfile to contain all
relevant paths to your input files. The "name" property of your reference and core species has to merely be a
unique identifier. It is however recommended to use whitespace-free species names to increase readability.Additional to the parameters file, you will need a tab separated file containing the position and sequence of each
miRNA for which a model should be constructed (more informationhere).You can then start CM construction with:ncCreate -p <parameters.yaml> -n <mirnas.tsv> -o <outdir>If you encounter errors, make sure that:The identifiers in the pairwise orthologs files match the ones in the gff files (use the-idtype=flag to use other
ID types)The contig/chromosome column in tab separated miRNA input file match the contig/chromosome id
in the reference gff fileUsencCreate -hto see all available options for CM construction.Orthology searchYou can start the orthology search with:ncSearch -m <CMs/> -n <mirnas.tsv> -q <query_genome.fa> -r <reference_genome.fa> -o <outdir>UsencSearch -hto see all available options for the orthology search or have a look at theWIKI.Phylogenetic AnalysisTo facilitate the downstream analyses of miRNA orthologs, we also supply thencAnalyzefunction:ncAnalzye -r <result directory of ncOrtho> -o <output_dir> -m <mappingfile>This will create a phylogenetic Profile ready for visualisation inPhyloProfileas well as calculate a supermatrix species tree based on the miRNA orthologs.More information can be found withncAnalyze -hor theWIKISupportPlease refer to our Wiki Page ofknown issuesfirst,
then consider opening an issue on GitHub or contacting me directly viamailContributorsFelix LangschiedAndreas BlaumeiserMirko BrüggemannDaniel AmselDept. for Applied Bioinformatics Institute for Cell Biology and Neurosciences, Goethe University, Frankfurt am MainIngo EbersbergerLicenseThis project is licensed under the GNU General Public License v3.0 - see theLICENSE.mdfile for detailsAcknowledgmentsLorenz et al. 2011: ViennaRNA Package 2.0Nawrocki et al. 2013: Infernal 1.1:
100-fold faster RNA homology searchesNotredame et al. 2000: T-Coffee: A Novel Method for Fast and
AccurateMultiple Sequence AlignmentShirley et al. 2015: Efficient "pythonic" access to FASTA files using pyfaidxContactFor support or bug reports please contact:langschied@bio.uni-frankfurt.de