motif_estimation.nf

params.bed = ""
params.out = ""
params.motif_db = ""
params.path_env = ""
params.help = 0
params.path_bin = "."

//bed_to_clustered_fasta
params.min_seq = 100  // Minimum number of sequences in the fasta-files for glam2

//glam2
params.motif_min_key = 8 // Minimum number of key positions (aligned columns)
params.motif_max_key = 20 // Maximum number of key positions (aligned columns)
params.iteration = 10000 // Number of Iterations done by glam2. A high iteration number equals a more accurate result but with an higher runtime.

//tomtom
params.tomtom_treshold = 0.01 // threshold for similarity score.

//cluster motifs
params.cluster_motif = 0 // Boolean if 1 motifs are clustered else they are not
params.edge_weight = 50 // Minimum weight of edges in motif-cluster-graph
motif_similarity_thresh = 0.00001 // threshold for motif similarity score

if (params.bed == "" || params.out == "" || params.motif_db == "" || params.path_env || "${params.help}" == "1") {
log.info """
Usage: nextflow run motif_estimation.nf --bed [PATH] --out [PATH] --motif_db [PATH] --path_env YAML-file
--bed Path			Input BED-file (Column with cluster: 11; column with sequenze: 10)!!
--out Path			Output Path
--motif_db Path		Path to MEME-Database
--path_env Path		Path to YAML file for conda enviroment.
--path_bin Path		Path to directory with subscripts
--help 0|1 Print help message if help == 1 (Default: 0)

Motif estimation:
--min_seq INT 		Sets the minimum number of sequences required for the FASTA-files given to GLAM2. (Default: 100)
--motif_min_key INT	Minimum number of key positions (aligned columns) in the alignment done by GLAM2. (Default: 8)
--motif_max_key INT	Maximum number of key positions (aligned columns) in the alignment done by GLAM2.f (Default: 20)
--iteration INT		Number of iterations done by glam2. More Iterations: better results, higher runtime. (Default: 10000)
--tomtom_treshold float	Threshold for similarity score. (Default: 0.01)
--best_motif INT	Get the best X motifs per cluster. (Default: 3)

Moitf clustering:
--cluster_motif	Boolean	If 1 pipeline clusters motifs. If its 0 it does not. (Defaul: 0)
--edge_weight INT	Minimum weight of edges in motif-cluster-graph (Default: 5)
--motif_similarity_thresh FLOAT	Threshold for motif similarity score (Default: 0.00001)
"""
System.exit(2)
} else {
	Channel.fromPath(params.bed).map {it -> [it.simpleName, it]}.set {bed_for_motif_esitmation}
}

/*
Converting BED-File to one FASTA-File per cluster
*/
process bed_to_clustered_fasta {
	conda "${params.path_env}"
	publishDir "${params.out}/esimated_motifs/clustered_motifs/clustered_fasta/", mode: 'copy'
    tag{name}

    input:
    set name, file (bed) from bed_for_motif_esitmation

    output:
    file ('*.FASTA') into fasta_for_glam2
    file ('*.FASTA') into fasta_for_motif_cluster

    script:
    """
    Rscript ${params.path_bin}/bed_to_fasta.R ${bed} ${name} ${params.min_seq}
    """
}


//flatten list and adding name of file to channel value
fasta_for_glam2 = fasta_for_glam2.flatten().map {it -> [it.simpleName, it]}
//combine fasta files in one list
fasta_for_motif_cluster = fasta_for_motif_cluster.toList()

/*
Running GLAM2 on FASTA-files.
Generating Motifs through alignment and scoring best local matches.
*/
process glam2 {

    tag{name}
	publishDir "${params.out}/esimated_motifs/clustered_motifs/${name}/", mode: 'copy'

    input:
    set name, file (fasta) from fasta_for_glam2

    output:
    file("${name}/*.meme") into meme_to_merge
	set name, file("${name}/*.meme") into meme_for_tomtom
	set name, file("${name}/*.meme") into meme_for_filter
	file ('*')

    script:
    """
    glam2 n ${fasta} -O ./${name}/ -a ${params.motif_min_key} -b ${params.motif_max_key} -z 5 -n ${params.iteration}
    """
}

/*
Combining all MEME-files to one big MEME-file.
The paths are sorted numerically depending on the cluster number.
*/
process merge_meme {

	publishDir "${params.out}/esimated_motifs/merged_meme/", mode: 'copy'

	input:
	val (memelist) from meme_to_merge.toList()

	output:
	file ('merged_meme.meme') into merged_meme

	when:
	params.cluster_motif == 1

	script:
	memes = memelist.collect{it.toString().replaceAll(/\/glam2.meme/,"") }
	meme_sorted = memes.sort(false) { it.toString().tokenize('_')[-1] as Integer }
	meme_sorted_full = meme_sorted.collect {it.toString() + "/glam2.meme"}
	meme_list = meme_sorted_full.toString().replaceAll(/\,|\[|\]/,"")
	"""
	meme2meme ${meme_list} > merged_meme.meme
	"""
}

/*
Running Tomtom on merged meme-files.
Output table has the information which clusters are similar to each other.
*/
process find_similar_motifs {

	publishDir "${params.out}/esimated_motifs/cluster_similarity/", mode: 'copy'
	input:
	file (merged_meme) from merged_meme

	output:
	file ('all_to_all.tsv') into motif_similarity

	when:
	params.cluster_motif == 1

	script:
	"""
	tomtom ${merged_meme} ${merged_meme} -thresh ${params.motif_similarity_thresh} -text --norc | sed '/^#/ d' | sed '/^\$/d' > all_to_all.tsv
	"""
}


files_for_merge_fasta = motif_similarity.combine(fasta_for_motif_cluster)


/*
Merging FASTA-files of similar clusters
*/
process merge_fasta {
	conda "${params.path_env}"
	publishDir "${params.out}/esimated_motifs/merged_fasta/", mode: 'copy'

	input:
	set file (motiv_sim), val (fasta_list) from files_for_merge_fasta

	output:
	file ('*.fasta') into motif_clustered_fasta_list
  	file('*.png')

  	when:
  	params.cluster_motif == 1

	script:
  	fa_sorted = fasta_list.sort(false) { it.getBaseName().tokenize('_')[-1] as Integer }
  	fastalist = fa_sorted.toString().replaceAll(/\s|\[|\]/,"")
	"""
	Rscript ${params.path_bin}/merge_similar_clusters.R ${motiv_sim} ${fastalist} ${params.edge_weight}
	"""
}


motif_clustered_fasta_flat = motif_clustered_fasta_list.flatten()


process clustered_glam2 {

	publishDir "${params.out}/final_esimated_motifs/${name}/", mode: 'copy'

	input:
	file (fasta) from motif_clustered_fasta_flat

	output:
	set name, file ('*.meme') into clustered_meme_for_tomtom
  	set name, file ('*.meme') into clustered_meme_for_filter
  	file('*')

	when:
	params.cluster_motif == 1

	script:
  	name = fasta.getBaseName()
	"""
	glam2 n ${fasta} -O . -a ${params.motif_min_key} -b ${params.motif_max_key} -z 5 -n ${params.iteration}
	"""
}

if(params.cluster_motif == 1){
	for_tomtom = clustered_meme_for_tomtom
	for_filter = clustered_meme_for_filter
} else {
	for_tomtom = meme_for_tomtom
	for_filter = meme_for_filter
}


/*
Running Tomtom on meme-files generated by GLAM2.
Tomtom searches motifs in databases.
*/
process tomtom {

    tag{name}
	publishDir "${params.out}/esimated_motifs/tomtom/", mode: 'copy'

    input:
    set name, file (meme) from for_tomtom

    output:
    set name, file ('*.tsv') into tsv_for_filter

    script:
    """
    tomtom ${meme} ${params.motif_db} -thresh ${params.tomtom_treshold} -mi 1 -text | sed '/^#/ d' | sed '/^\$/d' > ${name}_known_motif.tsv
    """
}


//Joining channels with meme and tsv files. Filter joined channel on line count.
//Only meme-files which corresponding tsv files have linecount <= 1 are writen to next channel.
for_filter2 = for_filter.join( tsv_for_filter )
for_filter2
    .filter { name, meme, tsv ->
        long count = tsv.readLines().size()
        count <= 1
        }
    .into { meme_for_scan; check }


//If channel 'check' is empty print errormessage
process check_for_unknown_motifs {
    echo true

    input:
    val x from check.ifEmpty('EMPTY')

    when:
    x == 'EMPTY'

    """
    echo '>>> STOPPED: No unknown Motifs were found.'
    """

}


//Get the best(first) Motif from each MEME-file
process get_best_motif {
	conda "${params.path_env}"

	publishDir "${params.out}/esimated_motifs/unknown_motifs/", mode: 'copy'

	input:
	set name, file(meme), file(tsv) from meme_for_scan

	output:
	set name, file('*_best.meme') into best_motif

	script:
	"""
	python ${params.path_bin}/get_best_motif.py ${meme} ${name}_best.meme ${params.best_motif}
	"""
}