Workflow Overview

graph LR;
    fq[FASTQ]
    ref[FASTA]
    jc["Intersect Variant Calls"]
    subgraph "Read Processing"
    fq -->|cutadapt|tfq["Trimmed Reads"]
    end
    subgraph Alignment
    tfq -->|bwa-mem|al["BAM"]
    ref --> al
    end
    subgraph "Variant Calling"
    al -->|freebayes|fb["Freebayes Calls"]
    al -->|lofreq|lf["LoFreq Calls"]
    fb -->|bcftools|jc
    lf -->|bcftools|jc
    end
    subgraph "Variant Filtering"
    jc -->|rustynuc|fjc["Joint Calls"]
    fjc --> hf{"Heatmap Filtering"} --> hv["Heatmap Variants"]
    end

Data

Raw reads are deposited on ENA and can be accessed using the link below:

PRJEB51942

Reference genome and annotation files can be obtained from the links below:

FASTA GFF3 GTF

Dependencies

The simplest way to obtain all the tools or dependencies for variant calling/filtering is from the bioconda channel.

Before being able to create the Conda environment you will need to install conda for your specific OS.

You can install Miniconda for your specific platform from HERE

Copy the content of the box below into a file named lowfreqvar.yml:

name: lowfreqvar
channels:
  - conda-forge
  - bioconda
  - defaults
dependencies:
  - bioconda::cutadapt=2.10
  - bioconda::bcftools=1.10.2
  - bioconda::bwa-mem=0.7.17
  - bioconda::freebayes=1.3.2
  - bioconda::lofreq=2.1.5
  - bioconda::rustynuc=0.3.0

to create the environment containing all the dependencies run:

conda env create -f lowfreqvar.yml

and to activate the environment containing all the tools run:

conda activate lowfreqvar