Reproducing the Analysis

Analysis was run in 3 different environments:

• HPC "compute nodes" with no internet access
• HPC "software node" with limited internet access
• laptop running MacOS with full internet access and access to storage space visible to HPC nodes

Setup

The following was run on a "software node":

# mamba forge was installed into home directory and sourced
curl -L -O "https://github.com/conda-forge/miniforge/releases/latest/download/Mambaforge-$(uname)-$(uname -m).sh"
bash Mambaforge-$(uname)-$(uname -m).sh
source ~/mambaforge/bin/activate

# environment was created from the pgt-pgs-markers/envs directory
git clone https://github.com/SaundersLab/pgt-pgs-markers.git
cd pgt-pgs-markers/envs
mamba env create -f pgt-pgs-markers-env.yml --prefix pgt-pgs-markers
cd ..

# installation of bwa 0.7.5 in the pgt-pgs-markers directory
curl -L -O https://github.com/lh3/bwa/archive/refs/tags/0.7.5.tar.gz
tar -xvf 0.7.5.tar.gz
cd bwa-0.7.5
make

The following was run on a laptop:

# mamba forge was installed into home directory and sourced
curl -L -O "https://github.com/conda-forge/miniforge/releases/latest/download/Mambaforge-$(uname)-$(uname -m).sh"
bash Mambaforge-$(uname)-$(uname -m).sh
source ~/mambaforge/bin/activate

# environment was created from the pgt-pgs-markers/envs directory
git clone https://github.com/SaundersLab/pgt-pgs-markers.git
cd pgt-pgs-markers/envs
mamba env create -f download-reads-env.yml --prefix download-reads-env
conda activate $PWD/download-reads-env
cd ..

path_to_hpc_storage_from_laptop=/path/to/hpc/storage/from/laptop

# script to download reads was run
./src/download_reads.sh "$path_to_hpc_storage_from_laptop"/pgt-pgs-markers/data/reads

The reference genome and annotations were downloaded from https://genome.jgi.doe.gov/portal/Pgt_201_B1/Pgt_201_B1.download.html (account required)

• Pgt_201_B1_GeneCatalog_20191015.gff3.gz
• Pgt_201_B1_AssemblyScaffolds2.fasta.gz

These were unzipped and copied into pgt-pgs-markers/data/reference

The empty lines (882866 and 1018814) were removed from Pgt_201_B1_AssemblyScaffolds2.fasta.

Alignments, Marker Finding, and Tree Building

The partitions variable was set to a comma separated list of partition names specific to the cluster.

mkdir -p logs
partitions=comma,separated,list,of,partition,names
sbatch -p $partitions src/analyse_samples.sh $partitions

Primer design

Results from results/markers.csv were used with Primer-BLAST.

The following changes were made from the default parameters:

Parameter	Default	Value Used
# of primers to return	10	30
Primer Size Max	25	30
GC clamp	0	1

The following assemblies were used (links below are to the Primer-BLAST page for each assembly):

Pgt assemblies:

• 21-0
• CRL 75-36-700-3
• Ug99
• UK-01
• 99KS76A-1

Wheat:

• Chinese Spring

Rye:

• Lo7

Primers were designed on the 21-0 assembly and then checked against all the other assemblies listed.

Analysis of chosen markers

# assuming we're still in the pgt-pgs-markers directory and 
# download-reads-env environment is still active
conda deactivate
cd envs
mamba env create -f analyse-chosen-markers.yml --prefix analyse-chosen-markers
conda activate $PWD/analyse-chosen-markers
cd ..

./src/analyse_chosen_markers.sh "$path_to_hpc_storage_from_laptop"/pgt-pgs-markers/results

Figures produced were then rearranged in a vector graphics editor.