VEME2021

Install UShER via conda

• Setup the channels

conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

• Install UShER package

conda install usher

• Update the package

conda update usher

Download UShER demo files

wget https://raw.githubusercontent.com/russcd/USHER_DEMO/master/example_files.zip
unzip example_files.zip

Convert FASTA files to VCF

• Download the reference file

wget https://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/bigZips/wuhCor1.fa.gz
gunzip wuhCor1.fa.gz

• Create an MSA of all sequences (including the reference)

mafft --thread 4 --auto --keeplength --addfragments example1_typical_use_case.fasta wuhCor1.fa > example_1_msa.fa

• Mask problematic sites

wget https://raw.githubusercontent.com/W-L/ProblematicSites_SARS-CoV2/master/problematic_sites_sarsCov2.vcf

• Generate VCF from MSA

faToVcf -maskSites=problematic_sites_sarsCov2.vcf example_1_msa.fa example_1.vcf

Place VCF sequences onto a comprehensive SARS-CoV-2 phylogenetic tree made up of public (GenBank, COG-UK and CNCB) sequences

• Download the comprehensive phylogenetic tree (dated 2021-08-25 in the example below and the subsequent commands)

wget http://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/2021/08/25/public-2021-08-25.all.masked.nextclade.pangolin.pb.gz

• Place the sequences on the comprhensive global tree using UShER

usher -i public-2021-08-25.all.masked.nextclade.pangolin.pb.gz -v example_1.vcf -o example_1_placed.pb -T 4 -k 50 -u -D

Files generated to view: uncondensed-final-tree.nh, mutation-paths.txt, clades.txt, subtree-1.nh, subtree-1-mutations.txt, example_1_placed.pb, etc.

Tutorials

Summarize the mutation-annotate tree (MAT)

• Basic summary

matUtils summary -i public-2021-08-25.all.masked.nextclade.pangolin.pb.gz -T 4
matUtils summary -i example_1_placed.pb -T 4

• Detailed summary

matUtils summary -i public-2021-08-25.all.masked.nextclade.pangolin.pb.gz -T 4 -A -d summary_out/

Files to view under the summary_out folder.

• Getting amino acid translation of mutations

wget http://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/bigZips/genes/ncbiGenes.gtf.gz
gunzip ncbiGenes.gtf.gz
matUtils summary -i public-2021-08-25.all.masked.nextclade.pangolin.pb.gz -T 4 -g ncbiGenes.gtf -f wuhCor1.fa -t coding_mutations.tsv

Extracting subtrees from a MAT to view in Auspice/Newick viewers

• Store the sequence names of the samples of interest in a new file (samples_of_interest.txt in the example below)

grep ">" example1_typical_use_case.fasta | cut -d">" -f2 > samples_of_interest.txt 

• Extract subtree of samples provided as a Newick file (samples_of_interest.nh in the example below)

matUtils extract -i example_1_placed.pb -T 4 -s samples_of_interest.txt -t samples_of_interest.nh 

• Extract subtree of samples provided as a JSON file (samples_of_interest.json in the example below)

matUtils extract -i example_1_placed.pb -T 4 -s samples_of_interest.txt -j samples_of_interest.json 

• Extract subtree(s) of size 50 in the context of the samples provided and output the JSON files

matUtils extract -i example_1_placed.pb -T 4 -K samples_of_interest.txt:50  

• Extract the clade corresponding to clade AY.2 and output the Newick and JSON files

matUtils extract -i example_1_placed.pb -T 4 -c "AY.2" -t AY2.nh -j AY2.json -o AY2.pb

Converting a MAT to be viewable in Taxodium

• Download metadata files for the comprehensive tree

wget https://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/2021/08/25/public-2021-08-25.metadata.tsv.gz
gunzip public-2021-08-25.metadata.tsv.gz

• Use the metadata to convert a MAT to taxodium pb file

matUtils extract -i public-2021-08-25.all.masked.nextclade.pangolin.pb.gz -l taxodium_public-2021-08-25.pb -M public-2021-08-25.metadata.tsv -g ncbiGenes.gtf -f wuhCor1.fa 

Optimize a MAT for parsimony

matOptimize -i AY2.pb -T4 -o AY2_optimized.pb

• To view the optimized MAT in Auspice

matUtils extract -i AY2_optimized.pb -T 4 -j AY2_optimized.json 

Check for recombination ancestry in the samples of interest

ripples -i example_1_placed.pb -T 4 -s samples_of_interest.txt 

Files to view: recombination.tsv and descendants.tsv

UShER Web Tool

• Upload example1_typical_use_case.fasta to https://genome.ucsc.edu/cgi-bin/hgPhyloPlace.

References

UShER:

• Yatish Turakhia, Bryan Thornlow, Angie S Hinrichs, Nicola de Maio, Landen Gozashti, Robert Lanfear, David Haussler, and Russ Corbett-Detig, "Ultrafast Sample placement on Existing tRees (UShER) enables real-time phylogenetics for the SARS-CoV-2 pandemic", Nature Genetics (2021) paper.

matUtils:

• Jakob McBroome*, Bryan Thornlow*, Angie S. Hinrichs, Nicola De Maio, Nick Goldman, David Haussler, Russell Corbett-Detig, Yatish Turakhia, "A daily-updated database and tools for comprehensive SARS-CoV-2 mutation-annotated trees", bioRxiv (2021), preprint

RIPPLES:

• Yatish Turakhia*, Bryan Thornlow*, Angie S. Hinrichs, Jakob McBroome, Nicolas Ayala, Cheng Ye, Nicola De Maio, David Haussler, Russell Corbett-Detig, "Pandemic-Scale Phylogenomics Reveals Elevated Recombination Rates in the SARS-CoV-2 Spike Region", bioRxiv (2021), preprint

Masking recomendations:

• Yatish Turakhia, Nicola De Maio, Bryan Thornlow, Landen Gozashti, Robert Lanfear, Conor R. Walker, Angie S. Hinrichs, Jason D. Fernandes, Rui Borges, Greg Slodkowicz, Lukas Weilguny, David Haussler, Nick Goldman and Russell Corbett-Detig, "Stability of SARS-CoV-2 Phylogenies", PLOS Genetics 2020 paper.

matOptimize

• Coming soon.

Taxodium

• Expected soon (check https://github.com/theosanderson/taxodium for updates).

Wiki

• https://usher-wiki.readthedocs.io/