Sars-CoV-2 Lineage Identification Workflow

A Snakemake workflow for identifying a Sars-CoV-2 lineage from a paired-end read. It implements both de-novo and reference-guided genome assembly, and uses Pangolin to identify the Covid lineage.

This project was built by group 3 for the DataScience in Bioinformatics WiSe 2022/23 course.

System Requirements

• Operating System: Recent version of Linux, Ubuntu 20.04 or newer is recommended
• Memory: At least 8GB
• CPU: At least 2 cores are recommended
• Storage: At least 10 GB of free space

Requirements can vary depending on the sample sizes and number of samples.

Installation

You can either run the program directly using Snakemake, or you can use a Docker image which has all of the software and libraries already installed.

Using Snakemake

Step 1: Install Mambaforge and Snakemake

With Python installed, follow the instructions found on the official Snakemake Installation guide to install Mambaforge and Snakemake.

Make sure to do a full installation, creating an environment named snakemake:

$ conda activate base
$ mamba create -c conda-forge -c bioconda -n snakemake snakemake

Step 2: Clone The Repository

Run the following command to clone the repository from GitHub:

$ git clone https://github.com/shahab-ab/project-g3.git

Or download the latest release and extract it on your machine.

Using Docker

Step 1: Install Docker Engine

From the Docker Engine installation page install the appropriate Docker Engine version for your system.

Step 2: Pull Docker Image

Run the following command to pull the latest image of the workflow from Docker Hub:

$ docker pull docksh360/project-g3:latest

Input

Copy the samples whose lineage you want to identify to data/samples (if you have not cloned the repository, create the directory data/samples yourself). Each sample must consist of a paired-end read. Sample files must be named as follows:

<SAMPLE_NAME><READ1_SUFFIX>.fastq.gz # First read
<SAMPLE_NAME><READ2_SUFFIX>.fastq.gz # Second read

Here is an example of a paired-end read of a sample named my_sample:

$ ls data/samples
my_sample.1.fastq.gz  my_sample.2.fastq.gz

Then edit config/config.yaml (if you have not cloned the repository, create config/config.yaml yourself) and change the samples object to reflect the names of the samples. The samples object contains one sub-object for each sample, whose key is the <SAMPLE_NAME> mentioned above, and which has two properties:

• r1_suffix: The suffix for the first read (<READ1_SUFFIX), without the .fastq.gz extension
• r2_suffix: The suffix for the second read (<READ2_SUFFIX), without the .fastq.gz extension

Here is a full example of config/config.yaml, which refers to two paired-end sample reads named my_sample.1.fastq.gz and my_sample.2.fastq.gz (in this case, my_sample is the <SAMPLE_NAME> and .1 and .2 are <READ1_SUFFIX> and <READ2_SUFFIX> respectively:

samples:
    "my_sample":
      r1_suffix: ".1"
      r2_suffix: ".2"

Running

With Snakemake

From the project's main directory, run the following commands:

$ conda activate snakemake # activate the snakemake environment if necessary
$ snakemake --use-conda --cores 4 report/qc/multiqc.html

You may change the number of cores (4 in this instance) to reflect how many cores you would like the workflow to make use of.

With Docker

From the project's main directory, run the following command:

$ docker run --rm \
	--volume="`pwd`/data/samples:/project/data/samples:ro" \
	--volume="`pwd`/config:/project/config:ro" \
	--volume="`pwd`/report/qc:/project/report/qc" \
	docksh360/project-g3:latest

Explanation: This command creates a temporary container using the docksh360/project-g3:latest image, mounts the required input and output directories as volumes, and runs the command to generate the final output.

Output

After successfully running the workflow, all of the results can be found in report/qc/multiqc.html. The most relevant section would be the Pangolin section, which shows a table containing entries named as follows:

• <SAMPLE_NAME>: The final result, obtained by selecting the better result between de-novo and reference-guided assembly
• [denovo] <SAMPLE_NAME>: The result obtained using de-novo assembly
• [refbased] <SAMPLE_NAME>: The result obtained using reference-guided assembly

This is an example output of running the workflow on 4 different samples:

In this example, RV417026_S15_L001 is identified assigned different lineages by de-novo and reference-guided assembly. However, we notice that conflict for the reference-guided result has a value of 0.1, whereas with de-novo, conflict is 0. In this case, we picked the result with the lower conflict value.

File Structure and Organization

The project has the following directory structure:

• config/config.yaml: Used to specify the names of the samples.
• data/genomes/genome.fa: The reference Sars-CoV-2 genome, NC_045512.2.
• data/samples/: Should contain the samples on which to run the workflow.
• workflow/envs/: Contains the Conda environments which are used by some of the tools in the workflow.
• workflow/rules/: Contains all of the rules for the workflow. All files directly under this directory are shared by both de-novo and reference-guided assignment.
• workflow/rules/de_novo/: Contains all of the rules that are specific to de-novo assembly.
• workflow/rules/reference_guided/: Contains all of the rules that are specific to reference-guided assembly.
• workflow/scripts/: Contains the scripts that are used in the workflow.

Additionally, the following directories are created upon running the workflow:

• logs/: Contains the output logs for all the rules.
• report/: Contains the final MultiQC report, along with reports from other tools.
• results/: Contains all of the intermediate files used by the rules.

Workflow

This following flowchart shows the main steps involved in this workflow. Please note that some steps are omitted for brevity. For the full list of steps, you can either generate a Snakemake report by running the command snakemake --report report.html, or read the source code.

Attribution

• Jean Qussa
• Shahab Abbaszadeh
• Yulin Luo

Resources

These are some of the resources that were directly or indirectly used in the creation of this workflow:

• https://www.ncbi.nlm.nih.gov/sra/docs/sars-cov-2-illumina-variant-calling-pipeline/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9013232/
• https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262953
• https://github.com/dridk/Sars-CoV-2-NGS-pipeline
• https://github.com/charlesfoster/covid-illumina-snakemake
• https://www.ridom.de/u/SARS-CoV-2_Analysis_Quick_Start.html