SYNOPSIS

    miRkwood is an application that allows for the fast and easy identification of microRNAs. It is specifically designed for plant microRNAs.


INSTALL

    See file miRkwood_installation.md.


USAGE

    miRkwood comes in two distinct pipelines, according to the input data type.

    -mirkwood.pl (abinitio pipeline): scans a genomic sequence and finds all potential microRNA precursors.
        Input: a FASTA file.

    -mirkwood-bed.pl (smallRNAseq pipeline): analyses small RNA deep sequencing data and find all potential microRNAs.
        Input : a BED file.


OPTIONS

    -mirkwood.pl: perl -I/{miRkwood_path}/cgi-bin/lib/ mirkwood.pl [options]
          Mandatory options:
            --input
                Path to the fasta file.

            --output
                Output directory. If non existing it will be created. The directory
                must be empty.

          Additional options:
            --both-strands
                Scan both strands.

            --species-mask
                Mask coding regions against the given organism.

            --shuffles
                Compute thermodynamic stability (shuffled sequences).

            --filter-mfei
                Select only sequences with MFEI < -0.6.

            --filter-rrna
                Filter out ribosomal RNAs (using RNAmmer).

            --filter-trna
                Filter out tRNAs (using tRNAscan-SE).

            --align
                Flag conserved mature miRNAs (alignment with miRBase + miRdup).

            --varna
                Allow the structure generation using Varna.

            --help
                Print a brief help message and exits.

            --man
                Prints the manual page and exits.


    -mirkwood-bed.pl: perl -I/{miRkwood_path}/cgi-bin/lib/ mirkwood-bed.pl [options]
          Mandatory options:
            --input
                Path to the BED file (created with our script mirkwood-bam2bed.pl).

            --genome
                Path to the genome (fasta format).

            --output
                Output directory. If non existing it will be created. The directory
                must be empty.

          Additional options:
            --shuffles
                Compute thermodynamic stability (shuffled sequences).

            --align
                Flag conserved mature miRNAs (alignment with miRBase + miRdup).

            --no-filter-mfei
                Don't filter out sequences with MFEI >= -0.6. Default : only keep
                sequences with MFEI < -0.6.

            --mirbase
                If you have a gff file containing known miRNAs for this assembly,
                use this option to give the path to this file.

            --gff
                List of annotation files (gff or gff3 format). Reads matching with
                an element of these files will be filtered out. For instance you can
                filter out CDS by providing a suitable GFF file.

            --no-filter-bad-hairpins
                By default the candidates with a quality score of 0 and no
                conservation are discarded from results and are stored in a BED
                file. Use this option to keep all results.

            --min-read-positions-nb
                Minimum number of positions for each read to be kept. Default : 0.

            --max-read-positions-nb
                Maximum number of positions for each read to be kept. Default : 5
                (reads that map at more than 5 positions are filtered out).

            --varna
                Allow the structure generation using Varna.

            --help
                Print a brief help message and exits.

            --man
                Prints the manual page and exits.


OUTPUT

    For both pipelines:

        alignments : folder containing all alignments files
            (only if option --align is on).

        images: folder containing images created by VARNA
            (only if option --varna is on).

        results: folder containing all results files, in several 
            formats (csv, fa, gff, html and txt).

        sequences: folder containing sequences for each candidate 
            in fasta and dotbracket format, alternatives sequences 
            if they exist and optimal structure if it is different 
            from the stemloop structure.

        YML: folder containing all candidates data in YAML format.

        basic_candidates.yml: contains a summary of all candidates
            with basic informations (this file is needed to create
            the results files).

        log.log: log file (hey, what did you expect?)

        run_options.cfg: config file with the chosen options.

    ab initio pipeline only:

        masks: folder containing results of BlastX, rnammer and tRNAscan-SE.

        input_sequences.fas: your sequences.

    smallRNAseq pipeline only:

        read_clouds: folder containing all text files for the candidates 
            read clouds.

        bed_sizes.txt: tabulated file with the number of reads in each BED file.

        summary.txt: contains a summary of your options and of results.


        Depending on the options you chose for your job you may find 
        some of the following files:

            your_bed_your_GFF.tar.gz: a compressed BED containing all reads matching
                to features from your GFF file, for each GFF file that you
                provided.

            your_bed_multimapped.tar.gz: a compressed BED containing all reads from your 
                input BED file mapping at less than --min-read-positions-nb positions
                or more than --max-read-positions-nb positions.

            your_bed_miRNAs.tar.gz: a compressed BED containing all reads from your 
                input BED file corresponding to miRNAs present in miRBase.

            your_bed_orphan_clusters.tar.gz: a compressed BED containing all reads from your 
                input BED file that fall into a peak but that don't correspond to
                a valid miRNA candidate.

            your_bed_orphan_hairpins.tar.gz: a compressed BED containing all candidates
                with a quality score of 0 and no conservation. By default 
                these candidates are excluded from final results, but you can
                change this behaviour with flag option --no-filter-bad-hairpins.

            your_bed_filtered.bed: a BED containing all reads from your 
                input BED file that have not been filtered out in one of the
                previous categories.