Nextstrain Template

This repository provides a comprehensive Nextstrain analysis of "your virus". You can choose to perform either a shorter run with specific proteins or a full genome run.

For those unfamiliar with Nextstrain or needing installation guidance, please refer to the Nextstrain documentation.

Table of Contents

• Prerequisites
• Nextstrain Environment
  • Installation
  • Enhancing the Analysis
• Repository Organization
  • Configuration Files
• Usage Examples
  • Running a Build
  • Visualizing the Build
• Ingest
  • Sequences
• Acknowledgments
• Contact

Prerequisites

Ensure you have the following installed:

• Python=3.8 or higher
• Micromamba or Conda
• Snakemake=7
• Nextstrain CLI

Nextstrain Environment

Install the Nextstrain environment by following these instructions.

Installation

1. Clone the repository:

   git clone [email protected]:hodcroftlab/template_nextstrain.git
   cd template_nextstrain

2. Install the Nextstrain environment:

   micromamba create -n nextstrain \
     --override-channels --strict-channel-priority \
     -c conda-forge -c bioconda --yes \
     augur auspice nextclade \
     snakemake=7 git ncbi-datasets-cli
   
   micromamba activate nextstrain

3. Update/install additional dependencies:

   sudo apt-get update
   sudo apt-get install -y unzip
   
   micromamba install -c conda-forge -c bioconda csvtk seqkit tsv-utils ipdb entrez-direct
   micromamba install -c conda-forge fuzzywuzzy python-dotenv ipykernel

Enhancing the Analysis

The data for this analysis is available from NCBI Virus. Instructions for downloading sequences are provided under Sequences.

Repository Organization

This repository includes the following directories and files:

• scripts: Custom Python scripts called by the snakefile.
• snakefile: The entire computational pipeline, managed using Snakemake. Snakemake documentation can be found here.
• ingest: Contains Python scripts and the snakefile for automatic downloading of <your_virus> sequences and metadata.
• <protein_xy>: Sequences and configuration files for the specific protein_xy run.
• whole_genome: Sequences and configuration files for the whole genome run.

Configuration Files

The config, protein_xy/config, and whole_genome/config directories contain necessary configuration files:

• config.yaml: Configuration file for setting parameters and options for the analysis
• colors.tsv: Color scheme
• geo_regions.tsv: Geographical locations
• lat_longs.tsv: Latitude data
• dropped_strains.txt: It will exclude these accessions during augur filter
• clades_genome.tsv: Manually Labeling Clades on a Nextstrain Tree (see documentation here)
• reference_sequence.gb: Reference sequence (add manually)
• auspice_config.json: Auspice configuration file - has to be in all data folders!

The reference sequence used is XYZ, accession number, sampled in 19XX.

Usage Examples

Running a Build

Activate the Nextstrain environment:

micromamba activate nextstrain

To perform a build, run:

snakemake --cores 9 all

For specific builds:

• protein_xy build:

snakemake auspice/<your_virus>_protein_xy.json --cores 9

• Whole genome build:

snakemake auspice/<your_virus>_whole-genome.json --cores 9

Visualizing the Build

To visualize the build, use Auspice:

auspice view --datasetDir auspice

To run two visualizations simultaneously, you may need to set the port:

export PORT=4001

Ingest

For more information on how to run the ingest, please refer to the README in the ingest folder.

Sequences

Sequences can be downloaded manually or automatically.

1. Manual Download: Visit NCBI Virus, search for <your_virus> or Taxid XXXXXX, and download the sequences.
2. Automated Download: The ingest functionality, included in the main snakefile, handles automatic downloading.

The ingest pipeline is based on the Nextstrain RSV ingest workflow. Running the ingest pipeline produces data/metadata.tsv and data/sequences.fasta.

Acknowledgments

• Nextstrain
• Auspice
• Snakemake
• Biopython
• Genbank
• NCBI

Contact

For questions or support, please contact [[email protected]].

Feel free to adjust the content according to your project's specifics.