JBrowse Setup and Genome Analysis Guide

This guide provides step-by-step instructions for setting up JBrowse on cowpox, monkeypox, variola, and vaccinia in the Poxviridae viral family for synteny analysis and annotation across MacOS, AWS, and Windows. It includes commands, explanations, and troubleshooting tips.

The link to our working example: https://parshawn.github.io/

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MacOS Setup

Pre-requisites

1. Install Homebrew:

   /bin/bash -c "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/HEAD/install.sh)"

2. Install required tools:

   brew install samtools htslib node wget apache2 minimap2
   npm install -g @jbrowse/cli

3. Start Apache:

   sudo apachectl start

4. Set environment variable for Apache root:

   export APACHE_ROOT='/usr/local/var/www'

Setup JBrowse

1. Create a temporary directory for setup:

   mkdir tmp && cd tmp

2. Create and configure JBrowse directory:

   jbrowse create jbrowse2_output
   sudo mv jbrowse2_output $APACHE_ROOT/jbrowse2
   sudo chown -R $(whoami) $APACHE_ROOT/jbrowse2

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AWS Setup

Pre-requisites

1. Update and install dependencies:

   sudo apt update
   sudo apt install wget curl apache2 minimap2 samtools

2. Install Node.js and JBrowse CLI:

   curl -fsSL https://fnm.vercel.app/install | bash
   source ~/.bashrc
   fnm use --install-if-missing 20
   npm install -g @jbrowse/cli

3. Start Apache:

   sudo service apache2 start

4. Set environment variable for Apache root:

   export APACHE_ROOT='/var/www/html'

Setup JBrowse

1. Create a temporary directory for setup:

   mkdir tmp && cd tmp

2. Create and configure JBrowse directory:

   jbrowse create jbrowse2_output
   sudo mv jbrowse2_output $APACHE_ROOT/jbrowse2
   sudo chown -R $(whoami) $APACHE_ROOT/jbrowse2

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Windows Setup

Pre-requisites

1. Install required tools:

   • Node.js
   • Git Bash
   • Apache Server (WAMP/XAMPP)
   • Samtools/HTSlib (via Conda or binaries)

2. Install JBrowse CLI:

   npm install -g @jbrowse/cli

3. Set the Apache root path (e.g., C:\xampp\htdocs).

Setup JBrowse

1. Create a temporary directory for setup:

   mkdir tmp && cd tmp

2. Create and configure JBrowse directory:

   jbrowse create jbrowse2_output
   mv jbrowse2_output C:/xampp/htdocs/jbrowse2

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Downloading and Indexing Genomic Data

Repeat the following steps for each virus:

Variola Virus

export FASTA_ROOT="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/859/885/GCF_000859885.1_ViralProj15197/"
wget $FASTA_ROOT/GCF_000859885.1_ViralProj15197_genomic.fna.gz
gunzip GCF_000859885.1_ViralProj15197_genomic.fna.gz
mv GCF_000859885.1_ViralProj15197_genomic.fna variola.fna
samtools faidx variola.fna

Monkeypox Virus

export FASTA_ROOT="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/023/516/015/GCA_023516015.3_ASM2351601v1/"
wget $FASTA_ROOT/GCA_023516015.3_ASM2351601v1_genomic.fna.gz
gunzip GCA_023516015.3_ASM2351601v1_genomic.fna.gz
mv GCA_023516015.3_ASM2351601v1_genomic.fna monkeypox.fna
samtools faidx monkeypox.fna

Cowpox and Vaccinia

curl -o cowpox_genome_ncbi.fa "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NC_003663.2&rettype=fasta&retmode=text"
samtools faidx cowpox_genome_ncbi.fa

curl -o vaccinia_genome_ncbi.fa "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NC_006998.1&rettype=fasta&retmode=text"
samtools faidx vaccinia_genome_ncbi.fa

B14R Gene

curl -o B14R_gene.fna "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NC_001611.1&seq_start=163090&seq_stop=163539&strand=1&rettype=fasta&retmode=text"
samtools faidx B14R_gene.fna

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Synteny Analysis

Generate PAF Files

minimap2 monkeypox.fna variola.fna > variola_vs_monkeypox.paf
minimap2 cowpox_genome_ncbi.fa variola.fna > variola_vs_cowpox.paf
minimap2 vaccinia_genome_ncbi.fa variola.fna > variola_vs_vaccinia.paf

Add Variola Assembly

sudo jbrowse add-assembly variola.fna --load copy -n variola --out /var/www/html/jbrowse2

Add Monkeypox Assembly

sudo jbrowse add-assembly monkeypox.fna --load copy -n monkeypox --out /var/www/html/jbrowse2

Add Cowpox Assembly

sudo jbrowse add-assembly cowpox_genome_ncbi.fa --load copy -n cowpox --out /var/www/html/jbrowse2

Add Vaccinia Assembly

sudo jbrowse add-assembly vaccinia_genome_ncbi.fa --load copy -n vaccinia --out /var/www/html/jbrowse2

Add B14R Gene Assembly

sudo jbrowse add-assembly B14R_gene.fna --load copy -n B14R --out /var/www/html/jbrowse2

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Add Tracks to JBrowse

jbrowse add-track variola_vs_monkeypox.paf --assemblyNames variola,monkeypox --load copy --out $APACHE_ROOT/jbrowse2 --force
jbrowse add-track variola_vs_cowpox.paf --assemblyNames variola,cowpox --load copy --out $APACHE_ROOT/jbrowse2 --force
jbrowse add-track variola_vs_vaccinia.paf --assemblyNames variola,vaccinia --load copy --out $APACHE_ROOT/jbrowse2 --force

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Add Annotations

Variola Annotation

curl -o variola.gff.gz "https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/859/885/GCF_000859885.1_ViralProj15197/GCF_000859885.1_ViralProj15197_genomic.gff.gz"
gunzip variola.gff.gz
sudo jbrowse sort-gff variola.gff > variola_sorted.gff
bgzip variola_sorted.gff
tabix variola_sorted.gff.gz
sudo jbrowse add-track variola_sorted.gff.gz --assemblyNames variola --out /var/www/html/jbrowse2 --load copy

Monkeypox Annotation

curl -o monkeypox.gff.gz "https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/023/516/015/GCA_023516015.3_ASM2351601v1/GCA_023516015.3_ASM2351601v1_genomic.gff.gz"
gunzip monkeypox.gff.gz
sudo jbrowse sort-gff monkeypox.gff > monkeypox_sorted.gff
bgzip monkeypox_sorted.gff
tabix monkeypox_sorted.gff.gz
sudo jbrowse add-track monkeypox_sorted.gff.gz --assemblyNames monkeypox --out /var/www/html/jbrowse2 --load copy

Cowpox Annotation

curl -o cowpox_genome.gff.gz "https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/839/185/GCF_000839185.1_ViralProj14174/GCF_000839185.1_ViralProj14174_genomic.gff.gz"
gunzip cowpox_genome.gff.gz
sudo jbrowse sort-gff cowpox_genome.gff > cowpox_genome_sorted.gff
bgzip cowpox_genome_sorted.gff
tabix cowpox_genome_sorted.gff.gz
sudo jbrowse add-track cowpox_genome_sorted.gff.gz --assemblyNames cowpox --out $APACHE_ROOT/jbrowse2 --load copy

Vaccinia Annotation

curl -o vaccinia_genome.gff.gz "https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/860/085/GCF_000860085.1_ViralProj15241/GCF_000860085.1_ViralProj15241_genomic.gff.gz"
gunzip vaccinia_genome.gff.gz
sudo jbrowse sort-gff vaccinia_genome.gff > vaccinia_genome_sorted.gff
bgzip vaccinia_genome_sorted.gff
tabix vaccinia_genome_sorted.gff.gz
sudo jbrowse add-track vaccinia_genome_sorted.gff.gz --assemblyNames vaccinia --out $APACHE_ROOT/jbrowse2 --load copy

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Final Steps

Index Everything

jbrowse text-index --out $APACHE_ROOT/jbrowse2 --force

Publish to GitHub Pages

1. Navigate to the local GitHub repository:

   cd ~/github-repo

2. Copy the JBrowse folder to the repository:

   cp -r /var/www/html/jbrowse2 ~/github-repo/

3. Commit and push changes:

   git add .
   git commit -m "Add JBrowse setup and tracks"
   git push origin main

4. Enable GitHub Pages via repository settings.

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Troubleshooting

1. Synteny Tracks Empty:

   • Ensure PAF files match assembly names in JBrowse.
   • Verify PAF file structure for valid alignment data.

2. Permission Issues:

   • Use sudo where necessary.
   • Ensure correct permissions for Apache's document root and JBrowse folder.

3. GitHub Not Displaying Changes:

   • Ensure changes are committed to the correct branch.
   • Clear browser cache or reload the GitHub Pages URL.

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This guide provides a comprehensive walkthrough for setting up JBrowse and performing synteny analysis. For additional help, contact your system administrator or refer to JBrowse documentation.