viridian_simulations_workflow

A workflow to simulate SARS-CoV-2 amplicons and reads for assembly with viridian.

Overview

A working pipeline to generate and assemble synthetic SARS-CoV-2 amplicons.

The main steps are outlined below:

• The pipeline begins by simulating neutral evolution from the SARS-CoV-2 reference sequence using PhastSim and a reference SARS-CoV-2 phylogeny.
• Varifier is then used to generate truth VCFs for each simulated assembly using the SARS-CoV-2 reference sequence.
• The primer sequences of the specified amplicon scheme are mapped to the simulated assemblies using bwa aln and the amplicon sequence extracted for each primer pair. There is then a check for mismatches between each primer and the simulated assembly. If at least one mismatch is present, PCR artifacts are simulated with equal probability: the amplicon sequencing coverage is reduced to 0 or the sequence that the primer has mapped to is reverted to the reference sequence. Random amplicon dropout is also simulated with a default probability of 0.001. The sequencing coverage of amplicons containing no artifacts is simulated using a normal distribution with mean 500 and standard deviation 20 and each amplicon is written to a FASTA file.
• Sequencing reads are simulated for each amplicon using the sequencing coverages determined in the previous step. Illumina reads are simulated using ART illumina and Nanopore reads are simulated using Badread.
• Illumina reads are then assembled using the Connor Lab artic nextflow pipeline and viridian workflow and Nanopore reads are assembled using Epi2me and viridian workflow.
• Assemblies generated with the Connor lab pipeline and Epi2me are then trimmed to remove all bases outside of the specified amplicon scheme.
• Covid-truth-eval is then used to generate TSV files summarising the assembly accuracy for each tool.

Installation

# download the repository
git clone https://github.com/iqbal-lab-org/viridian_simulations_workflow
cd viridian_simulations_workflow
# install the python dependencies in a virtual env
python3 -m venv venv
source venv/bin/activate
pip3 install -r requirements.txt
# build the containers
python3 singularity/build_images.py

Usage

snakemake --cores <threads>

The pipeline takes a SARS-CoV-2 phylogeny titled "newick_output.nwk" as input, from which assemblies are simulated relative to a reference.

Configuration

General

• threads: Number of threads to extract amplicon sequences and run ART_illumina.
• reference_genome: A SARS-CoV-2 reference genome for simulations and read mapping.
• primer_scheme: The artic nCoV-2019 primer scheme (V3, V4 or V4.1).
• scheme_dir: GitHub repository of primer schemes for the artic assembly pipeline (usually https://github.com/artic-network/primer-schemes).
• seed: Start seed.
• container_directory: Path to the directory containing the singularity images.
• nextflow_path: Path to nextflow installation.

phastSim:

• tree_file: Newick tree of SARS-CoV-2 simulated genomes.
• output_dir: Output directory name for phastSim
• rate_parameter: Substitution rate for phastSim to use.

split_amplicons:

• random_dropout_probability: Probability of random dropout of amplicons to simulate PCR errors.
• primer_dimer_probability: Probability of amplicon being replaced by a primer dimer if either primer has a 3 base pair overlap with any other primer in the same PCR pool.
• match_coverage_mean: Mean coverage for amplicons containing no mismatches in the primer-binding region.
• match_coverage_sd: Standard deviation of coverage for amplicons containing no mismatches in the primer-binding region.
• mismatch_coverage_mean: Mean coverage for amplicons containing mismatches in the primer-binding region.
• mismatch_coverage_sd: Standard deviation of coverage for amplicons containing mismatches in the primer-binding region.

simulate_reads:

• illumina_read_length: Length of ART Illumina reads.

mask_assemblies:

• apply_mask: Mask low coverage bases in the simulated truth genomes.

artic_assemble

• scheme_url: URL to the GitHub repository containing the primer scheme specification.
• main_nf: Path to the Epi2me main nextflow file.
• illumina_workflow_container: Path to the artic illumina singularity container.

epi2me_badread_assemble

• main_nf: Path to the Epi2me main nextflow file.
• nxf_sing_cache: Directory for the nextflox cache.

viridian_assemble:

• viridian_container: Path to the viridian workflow singularity container.

Testing

To test the functions in scripts/error_modes.py, run python tests/run_tests.py