masterJLU2018

De novo motif discovery and evaluation based on footprints identified by TOBIAS.

For further information read the documentation.

Dependencies

Installation

Start with installing all dependencies listed above (Nextflow, conda, MEME-Suite) and downloading all files from the GitHub repository.
It is required to set the environment paths for meme-suite. this can be done with following commands:

export PATH=[meme-suite instalation path]/libexec/meme-[meme-suite version]:$PATH
export PATH=[meme-suite instalation path]/bin:$PATH

Every other dependency will be automatically installed using conda. For that a conda environment has to be created from the yaml-file given in this repository. It is required to create and activate the environment from the yaml-file beforehand. This can be done with following commands:

conda env create -f masterenv.yml
conda activate masterenv

Set the wd parameter in the nextflow.config file as path where the repository is saved. For example: '~/masterJLU2018/'.

Important Notes:

For conda the channel bioconda needs to be set as highest priority! This is required due to two different packages with the same name in different channels. For the pipeline the package jellyfish from the channel bioconda is needed and NOT the jellyfish package from the channel conda-forge!

Quick Start

nextflow run pipeline.nf --bigwig [BigWig-file] --bed [BED-file] --genome_fasta [FASTA-file] --motif_db [MEME-file] --organism [mm10|mm9|hg19|hg38]

Demo run

There are files provided inside ./demo/ for a demo run. Go to the main directory and run following command:

nextflow run pipeline.nf --bigwig ./demo/buenrostro50k_chr1_fp.bw --bed ./demo/buenrostro50k_chr1_peaks.bed --genome_fasta ./demo/hg38_chr1.fa --motif_db ./demo/jaspar_vertebrates.meme --out ./demo/buenrostro50k_chr1_out/ --organism hg38

Parameters

For a detailed overview for all parameters follow this link.

Required arguments:
	--bigwig		 Path to BigWig-file
	--bed			 Path to BED-file
	--genome_fasta		 Path to genome in FASTA-format
	--motif_db		 Path to motif-database in MEME-format
	--config		 Path to UROPA configuration file
 	--organism 		 Input organism [hg38 | hg19 | mm9 | mm10]
	--out			 Output Directory (Default: './out/')

Optional arguments:

	--help [0|1]		1 to show this help message. (Default: 0)
	--gtf_path		Path to gtf-file. If path is set the process which creates a gtf-file is skipped.
	--tfbs_path 		Path to directory with output from tfbsscan. If given tfbsscan will be skipped.

	Footprint extraction:
	--window_length INT	This parameter sets the length of a sliding window. (Default: 200)
	--step INT		This parameter sets the number of positions to slide the window forward. (Default: 100)
	--percentage INT	Threshold in percent (Default: 0)
	--min_gap INT		If footprints are less than X bases apart the footprints will be merged (Default: 6)

	Filter motifs:
	--min_size_fp INT	Minimum sequence length threshold. Smaller sequences are discarded. (Default: 10)
	--max_size_fp INT	Maximum sequence length threshold. Discards all sequences longer than this value. (Default: 200)
	--tfbsscan_method [moods|fimo] Method used by tfbsscan. (Default: moods)

	Cluster:
	Sequence preparation/ reduction:
	--kmer INT		K-mer length (Default: 10)
	--aprox_motif_len INT	Motif length (Default: 10)
	--motif_occurence FLOAT	Percentage of motifs over all sequences. Use 1 (Default) to assume every sequence contains a motif.
	--min_seq_length Interations	Remove all sequences below this value. (Default: 10)
	Clustering:
	--global INT		Global (=1) or local (=0) alignment. (Default: 0)
	--identity FLOAT	Identity threshold. (Default: 0.8)
	--sequence_coverage INT	Minimum aligned nucleotides on both sequences. (Default: 8)
	--memory INT		Memory limit in MB. 0 for unlimited. (Default: 800)
	--throw_away_seq INT	Remove all sequences equal or below this length before clustering. (Default: 9)
	--strand INT		Align +/+ & +/- (= 1). Or align only +/+ (= 0). (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. (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)
	--gap_penalty INT	Set penalty for gaps in GLAM2 (Default: 1000)
	Moitf clustering:
	--cluster_motif	Boolean	If 1 pipeline clusters motifs. If its 0 it does not. (Default: 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)

	Creating GTF:
	--tissues List/String 	List of one or more keywords for tissue-/category-activity, categories must be specified as in JSON
				config
All arguments can be set in the configuration files