AptaFlow for HT-SELEX NGS Data

This is the bioinformatic pipeline used for the Analysis of Enterococcus faecalis, performed in 2020 at TU Wien, Austria.

Workflow Diagram

Compact workflow: Workflow Diagram

Detailed workflow: Workflow Diagram

Getting Started

To replicate the results for the HT-SELEX against E. faecalis (EF05) the config-file ef05.config in the config folder can be used. Please note that in the config file the path to the raw NGS files has to be changed first.

Prerequisites & Installation

The workflow has been developed and run in an Anaconda environment. The conda environment is used for management of dependencies.

First of all, be sure to have at least Python 3.6 installed and install conda:

# Installing conda
pip install conda

# Updating conda base environment from default channel
conda update -n base -c defaults conda

# Adding of necessary channels
conda config --add channels bioconda
conda config --add channels conda-forge
conda config --add channels r

Next a conda environment, in which the dependencies will be installed, is created.

# Creating conda environment
conda create --name aptaflow

Activate the environment and install the dependencies.

conda activate aptaflow
conda install -c bioconda -c conda-forge nextflow=19.10.0 cutadapt=2.4 fastp=0.20.0 multiqc=1.9 pandas=1.1.0
conda install -c r r-base=3.6.1 r-dplyr=0.8.0.1 r-tidyr=0.8.3 r-ggplot2=3.1.1 r-xlsx=0.6.1 r-argparse=2.0.1 r-showtext=0.9

Packages used:

nextflow 19.10.0.5170
cutadapt 2.4
fastp 0.20.0
multiqc 1.9
pandas 1.1.0
dplyr 0.8.0.1
r-base 3.6.1
r-tidyr 0.8.3
r-ggplot2 3.1.1
r-xlsx 0.6.1
r-argparse 2.0.1
r-showtext 0.9

We had problems using the workflow on a fresh install of Linux (OpenSUSE) due to the xlsx-package using rJava. For it to properly work the environment variable LD_LIBRARY_PATH has to be set.

export LD_LIBRARY_PATH='$JAVA_HOME/lib:$JAVA_HOME/lib/server';

Be sure that $JAVA_HOME is pointing at your java install directory. You can check by echoing the $JAVA_HOME global variable:

echo $JAVA_HOME

Running the workflow

Make sure that the scripts in bin/ are executable. These scripts are called from within the aptaflow.nf script.

If they are not executable, use this command to make them executable.

chmod +x bin/*

Execute the pipeline like this:

nextflow run aptaflow.nf -c configs/YOUR_CONFIG.config

Or like this:

./aptaflow.nf run -c configs/YOUR_CONFIG.config

Config Files

Make sure to stick to the config file below and change only necessary variables, such as the round names, data location, random region length and so on.

There are also the config files used in our research for reproducibility.

// Parameters for nextflow script aptaflow.nf
params {

    // Provide a selex name for the output directory
    selex_name = "EF05"

    // provide rounds in sorted order
    rounds=["R00", "R02", "R03", "R04", "R05", "R06", "R07", "R08", "R09", "R10", "R11"]

    // random region length
    n=40

    // I/O
    input {
        raw_reads = 'data/*{R1,R2}_001.fastq'
        fwd_suffix="_L001_R1_001.fastq"
        rev_suffix="_L001_R2_001.fastq"
        round_delimiter = "_" // Will be cleaved e.g. R0_XYZ.fastq -> R0
    }
    output {
        out_dir = null
    }

    // System Specs
    specs {
        cpus = 24
    }

    // Primers
    primers {
        p5_f="TAGGGAAGAGAAGGACATATGAT"
        p3_f="TTGACTAGTACATGACCACTTGA"
        p5_r="TCAAGTGGTCATGTACTAGTCAA"
        p3_r="ATCATATGTCCTTCTCTTCCCTA"
    }

    // Trimming
    cutadapt {
        action = "trim"
        max_error = 0.2
        check_primer_contamination = false
        primer_contamination {
            max_error = 0.1
            min_overlap = 12
        }
        N = 40
        primer_length = 23
        max_deviation = 3
    }

    // Filtering and Merging
    fastp {
        filter_min_phred = 30    
    }
    
    // ViennaRNA (Currently not activated)
    vienna_rna {
    	T=21
    	mathews2004="VIENNA_RNA/misc/dna_mathews2004.par"
        for_top=10 // percent [0.00 - 100.00] of every round
    }
}

Affiliated research paper

Research paper associated with this GitHub Page.

License

No licensing agreement here yet.

vdejager/aptaflow