MycoSNP: GATK Variants

Overview

This repository contains the MycoSNP GATK Variants workflow, which consists of 10 steps:

1. Call variants using the GATK 4.1.4.1 HaplotypeCaller tool.
2. Combine gVCF files from the HaplotypeCaller into a single VCF using the GATK 4.1.4.1 CombineGVCFs tool.
3. Call genotypes using the GATK 4.1.4.1 GenotypeGVCFs tool.
4. Filter the variants using the GATK 4.1.4.1 VariantFiltration tool and the default (but customizable) filter: 'QD < 2.0 || FS > 60.0 || MQ < 40.0 || DP < 10'.
5. Run a customized VCF filtering script provided by the Broad Institute.
6. Split the filtered VCF file by sample.
7. Select only SNPs from the VCF files using the GATK 4.1.4.1 SelectVariants tool.
8. Split the VCF file with SNPs by sample.
9. Create a consensus sequence for each sample using BCFTools 1.9 and SeqTK 1.2.
10. Create a multi-fasta file from the VCF SNP positions using a custom script from Broad.

Requirements

Before installing and running this workflow, follow the instructions in the main repository to install the requirements: https://github.com/CDCgov/mycosnp

Installation

First install GeneFlow and its dependencies as follows:

1. Activate the previously installed Python virtual environment.

   cd ~/mycosnp
   source gfpy/bin/activate

2. Clone and install the mycosnp-gatk-variants workflow.

   gf install-workflow --make-apps -g https://github.com/CDCgov/mycosnp-gatk-variants mycosnp-gatk-variants

   The workflow should now be installed in the ~/mycosnp/mycosnp-gatk-variants directory.

   Note: To update the workflow, run the install-workflow command with the -f option as follows:

   gf install-workflow --make-apps -f -g https://github.com/CDCgov/mycosnp-gatk-variants mycosnp-gatk-variants

Execution

View the workflow parameter requirements using GeneFlow's help command:

cd ~/mycosnp
gf help mycosnp-gatk-variants

Execute the workflow with a command similar to the following. Be sure to replace the /path/to/indexed/reference with your indexed reference created using the MycoSNP BWA Reference workflow, and replace the /path/to/bam/index with the path to the indexed BAM files created by the MycoSNP BWA Pre-Process workflow:

gf --log-level debug run mycosnp-gatk-variants \
    -o ./output \
    -n test-mycosnp-gatk-variants \
    --in.input_folder /path/to/bam/index \
    --in.reference_sequence /path/to/indexed/reference \
    --param.max_perc_amb_samples 10 \
    --param.pair_hmm_threads 8

You can test this command out with the test data that was installed with the workflow as follows:

gf --log-level debug run mycosnp-gatk-variants \
    -o ./output \
    -n test-mycosnp-gatk-variants \
    --in.input_folder ~/mycosnp/mycosnp-gatk-variants/data/bam_index \
    --in.reference_sequence ~/mycosnp/mycosnp-gatk-variants/data/indexed_reference \
    --param.max_perc_amb_samples 10 \
    --param.pair_hmm_threads 8

Alternatively, to execute the workflow on an HPC system, you must first set the DRMAA library path environment variable. For example:

export DRMAA_LIBRARY_PATH=/opt/sge/lib/lx-amd64/libdrmaa2.so

Note that the DRMAA library for your specific scheduler (either UGE/SGE or SLURM) must be installed, and the installed library path may be different in your environment. Once the environment has been configured, execute the workflow as follows:

gf --log-level debug run mycosnp-gatk-variants \
    -o ./output \
    -n test-mycosnp-gatk-variants \
    --in.input_folder /path/to/bam/index \
    --in.reference_sequence /path/to/indexed/reference \
    --param.max_perc_amb_samples 10 \
    --param.pair_hmm_threads 8 \
    --ec default:slurm \
    --ep \
        default.slots:8 \
        'default.init:echo `hostname` && mkdir -p $HOME/tmp && export TMPDIR=$HOME/tmp && export _JAVA_OPTIONS=-Djava.io.tmpdir=$HOME/tmp && export PATH=/usr/sbin:$PATH && export XDG_RUNTIME_DIR=' \

Arguments are explained below:

1. -o ./output: The workflow's output will be placed in the ./output folder. This folder will be created if it doesn't already exist.
2. -n test-mycosnp-gatk-variants: This is the name of the workflow job. A sub-folder with the name test-mycosnp-gatk-variants will be created in ./output for the workflow output.
3. --in.input_folder: This must point to an output folder created by the MycoSNP BWA Pre-Process workflow, and should contain a sub-folder for each sample. Each sub-folder must contain a BAM file and a BAM index file (.bai)
4. --in.reference_sequence: This must point to an output folder created by the MycoSNP BWA Reference workflow, and should contain a reference sequence (FASTA), a .dict file, and a refererence sequence index (.fai)
5. --param.max_perc_amb_samples: Maximum percent of ambiguous samples allowed for inclusion of SNP in FASTA.
6. --param.pair_hmm_threads 8: Number of threads to use for GATK's variant calling algorithm. Recommended threads is 8.
7. --ec default:slurm: This is the workflow "execution context", which specifies where the workflow will be executed. "gridengine" or "slurm" is recommended, as this will execute the workflow on the HPC. However, "local" may also be used.
8. --ep: This specifies one or more workflow "execution parameters". a. default.slots:8: This specifies the number of CPUs or "slots" to request from the gridengine HPC when executing the workflow. The recommended number of slots is 8, and should match the pair_hmm_threads parameter. b. 'default.init:echo `hostname` && mkdir -p $HOME/tmp && export TMPDIR=$HOME/tmp && export _JAVA_OPTIONS=-Djava.io.tmpdir=$HOME/tmp && export XDG_RUNTIME_DIR=': This specifies a number of commands to execute on each HPC node to prepare that node for execution. This commands ensure that a local "tmp" directory is used (rather than /tmp), and also resets an environment variable that may interfere with correct execution of singularity containers.

After successful execution, the output directory should contain the following structure:

├── vcf-filter
│   ├── vcf-filter
│   └── _log
├── split-vcf-broad
│   ├── split-vcf-broad
│   └── _log
├── gatk-selectvariants
│   ├── gatk-selectvariants
│   └── _log
├── split-vcf-selectvariants
│   ├── split-vcf-selectvariants
│   └── _log
├── consensus
│   ├── consensus
│   └── _log
└── vcf-to-fasta
    ├── _log
    └── vcf-to-fasta

The consensus folder contains each consensus sequence FASTA in separate files. The vcf-to-fasta folder contains a multi-fasta file with only SNP positions. The gatk-selectvariants folder contains a VCF file with all samples, and only SNP positions. And the vcf-filter folder contains a VCF file filtered using a custom script provided by Broad. The split-vcf-* folders contain VCF files split by sample for each of the filtering steps.

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