OVERVIEW

RBRC is a tool for NGS paired-end read clustering and de novo assembly based on Reference genome sequence-based read clustering

REQUIREMENTS

System requirements

• gcc, g++ (7.5.0)
• make (GNU Make 4.1)
• java (1.8.0)
• wget (1.17.1)
• zip (3.0)
• git (2.7.4)

Perl libraries

• Sort::Key::Natural (perl library)
• Bio::TreeIO (perl library)
• Parallel::ForkManager (perl library)
• Switch (perl library)

INSTALLATION

Manual installation

• Download and install using RBRC package from this github page. You can install all third party tools automatically for running RBRC using 'setup.pl'.

    git clone https://github.com/jkimlab/RBRC.git
    cd RBRC
    ./setup.pl --install
  

Docker installation

• If you use Docker, you can download and use RBRC with all dependencies through pulling docker image.

    docker pull jkimlab/rbrc
  

• If you want to see manual for running RBRC docker image, see 👉 RBRC Docker hub

RUNNING RBRC

Example data

    ./setup.pl --example
    bash example_cmd.sh

    * Before running this command, you have to set the examples of RBRC

• Options of running RBRC

    ./RBRC.pl -p [parameter file] -o [output directory]
  

• To run RBRC, you need to prepare a parameter file as follows

    #---------------------------------------------------------------------------------------#
    ## Mendatory !
    # Reference genomes
    REF	1	<Reference name 1> <Reference genome sequence 1>
    REF	2	<Reference name 2> <Reference genome sequence 2>
  
    # NGS reads
    # FASTQ
    >LIB1
    <F read fastq file>
    <R read fastq file>
  
    #---------------------------------------------------------------------------------------#
    ## Optional
    # Running paramters
    THREADS        <number of threads: default = 1>
    REF_SIMILARITY_CUTOFF <minimum cutoff value of properly mapped reads: default = 80>
    MAPQ <read mapping quality threshold: default = 0>
  
    # Pairwise alignment & synteny block construction params
    RESOLUTION	<Resolution to construct synteny: default = 10000
  
    # Physical coverage paramters
    PHY_CUTOFF	LIB1	<minimum cutoff value for physical coverage-based syntenic region break: default = 5>
  
    # Distance based clustering paramters
    DBC_READ_DIST_CUTOFF	<maximum cutoff value of read distance for matrix calculation: default = 1000>
  
    # Cluster merging parameter
    MERGE_MIN_READS	<minimum cutoff value of links to merge clusters: default = 5>
    #---------------------------------------------------------------------------------------#
  

RBRC output

• Clustering output

    output_directory/RBRC.cluster : list of cluster and clustered reads
     - Column 1: name of cluster
     - Column 2: Read ID
     
    [example]
            CLUSTER1	chr8-278460/1
            CLUSTER1	chr8-278460/2
            CLUSTER1	chr8-278414/1
            CLUSTER1	chr8-278414/2
            CLUSTER1	chr8-278396/1
            CLUSTER1	chr8-278396/2
            CLUSTER1	chr8-278392/1
            CLUSTER1	chr8-278392/2
            CLUSTER1	chr8-278382/1
            CLUSTER1	chr8-278382/2
  

• Assembly output

    output_directory/SPAdes/Final_assembly/assembly.fasta
  

Required resourses for example datasets

Time

Coverage	2REF	3REF	4REF
5x	89 min	106 min	178 min
10x	85 min	91 min	103 min
30x	176 min	182 min	233 min
50x	279 min	285 min	370 min

Memory

Coverage	2REF	3REF	4REF
5x	1.26GB	1.38GB	1.51GB
10x	1.51GB	1.64GB	1.51GB
30x	4.28GB	4.28GB	4.28GB
50x	7.55GB	7.55GB	7.31GB

Disk

Coverage	2REF	3REF	4REF
5x	2.2G	2.4G	3.1G
10x	2.8G	2.9G	3.3G
30x	6.7G	7.2G	7.9G
50x	11G	12G	13G