Variants on protein structures part 2: degenerative myelopathy in dogs

JBrowse v5 beta

The tutorials target the JBrowse v5 beta, and the v4.3.0 release on the download page lacks some of what they show. To install the beta of JBrowse Web, run npm install -g @jbrowse/cli@next, then jbrowse create jbrowse2 --branch v5.0.0-beta.20. Desktop beta builds for Linux and macOS are on the releases page; the Windows build waits on code signing.

Degenerative myelopathy is a late-onset paralysis of dogs that resembles amyotrophic lateral sclerosis in people. A genome-wide association study found the risk allele in SOD1: c.118G>A changes glutamate 40 of superoxide dismutase to lysine (E40K), and most affected dogs are homozygous for it (Awano et al. 2009). We put the codon beside a crystal structure of the E40K protein itself, then read the allele across the Dog10K collection, with wolves and coyotes as the control, since an allele they share would predate domestication. We:

launch structure 7WX0 and an ortholog alignment from the SOD1 gene's right-click menu on the canFam4 dog assembly, with Launch protein view and Launch MSA view
slice SOD1 out of the Dog10K SNV callset with bcftools, and write a sample sheet of each animal's category
add the slice as a variant track, one row per animal, with the rows colored by category
open 7WX0 on the E40K record from a session spec, with the protein view's variant key labelling the change on the gene and the structure

Prerequisites

for your own JBrowse, the protein3d and msaview plugins from the plugin store; genomes.jbrowse.org already loads them
bcftools built with libcurl, and htslib (tabix)
curl

# Debian/Ubuntu
sudo apt install bcftools tabix
# or conda, which has every tool above
conda install -c bioconda -c conda-forge bcftools htslib

The data

The callset and the sample table come from the Dog10K consortium's public share (Meadows et al. 2023). You don't need these files to follow the page: the track reads hosted copies of what the commands below write. The slicing command streams the callset over HTTP, so a tabix-indexed VCF of your own slices the same way.

What it is	File
The SNV callset, 397 GB over 1,987 canids on canFam4 (UU_Cfam_GSD_1.0), streamed and never downloaded	AutoAndXPAR.SNPs.vqsr99.vcf.gz
The sample table, breed and category per animal	dog10K-alignment-sample-table.2022-02-23-v7.txt

Read by URL (no download needed)

What it is	File
Canine SOD1 E40K, PDB 7WX0	7WX0.cif
NCBI's ortholog report, the rows of the alignment	rest-api/

Opening SOD1 on codon 40 with the E40K structure

Part 1 opened its gene from the protein browser, which opens dog genes on a Labrador assembly. Dog10K is called against canFam4, so here we launch the same three views from the gene's right-click menu on canFam4. Codon 40 of NM_001003035.1 sits in SOD1's second coding exon, at chr31:27,123,057-27,123,059. Open the canFam4 hub on genomes.jbrowse.org and:

type chr31:27123013-27123103 into the location box
in the track selector, tick NCBI RefSeq - RefSeq Curated
right-click the SOD1 transcript and choose Launch protein view. On the PDB search tab pick 7WX0, then choose Open side by side from the arrow beside Launch
right-click the transcript again and choose Launch MSA view, which opens on its Orthologs tab; press Submit
click Reset Zoom, the circular arrow at the top right of the structure, to fit the whole structure
hover column 40 of the dog_query row in the alignment

The hover lights the codon on the gene, the column in the alignment and the residue on the structure, and the protein view's header compares the two letters: glutamate in the dog genome, lysine in the crystal.

SOD1 at codon 40 with its ortholog alignment and 7WX0. The hovered column, the codon band and Lys40 mark one position. Open this view in JBrowse ↗ (large dataset — this session takes a while to load)

Slicing SOD1 out of Dog10K

bcftools reads one gene of the tabix-indexed callset over HTTP and writes a small VCF of its genotypes. Set SNVS to your own callset's path or URL to slice it the same way:

SNVS=https://kiddlabshare.med.umich.edu/dog10K/SNP_and_indel_calls_2021-10-17/AutoAndXPAR.SNPs.vqsr99.vcf.gz
# -x INFO,^FORMAT/GT: drop every INFO field and every FORMAT field but GT
bcftools annotate -r chr31:27118000-27128000 -x INFO,^FORMAT/GT \
  -Oz -o dog10k_sod1.vcf.gz "$SNVS"
tabix -p vcf dog10k_sod1.vcf.gz

The sample table files each animal under a category:

curl -o sample-table.txt https://kiddlabshare.med.umich.edu/dog10K/sample-information/dog10K-alignment-sample-table.2022-02-23-v7.txt

JBrowse reads a sample sheet as a TSV whose first column, name, matches the VCF's sample names, with any further columns as metadata the rows can be colored by. An awk command writes that sheet from the table, with wolves first, then coyotes, village dogs, mixed and other dogs, and breed dogs, sorted by breed within each:

awk -F'\t' 'BEGIN { OFS = "\t"
    n = split("Wolf Coyote Village_Dogs Mixed/Other Breed_Dogs", o, " ")
    for (i = 1; i <= n; i++) rank[o[i]] = i }
  NR > 1 { print rank[$3], $1, $3, $2 }' sample-table.txt \
  | sort -t$'\t' -k1,1n -k4,4 -k2,2 | cut -f2- \
  | (printf 'name\tcategory\tbreed\n'; cat) > samples.tsv

E40K across breeds, village dogs and wild canids

The track reads hosted copies of the slice and the sheet; for your own, point vcfGzLocation and samplesTsvLocation at the files the commands wrote. rows.color names the sheet's column that colors each row:

Config file

Goes in the tracks array of config.json. See Tracks.

{
  "type": "VariantTrack",
  "trackId": "dog10k_sod1",
  "name": "Dog10K SNVs at SOD1",
  "assemblyNames": ["canFam4"],
  "adapter": {
    "type": "VcfTabixAdapter",
    "vcfGzLocation": {
      "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_sod1.vcf.gz"
    },
    "index": {
      "location": {
        "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_sod1.vcf.gz.tbi"
      }
    },
    "samplesTsvLocation": {
      "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_samples.tsv"
    }
  },
  "displays": [
    {
      "type": "LinearVariantDisplay",
      "displayId": "dog10k_sod1-LinearVariantDisplay",
      "rows": { "color": "category" }
    }
  ]
}

CLI (add-track-json)

jbrowse add-track-json '{
  "type": "VariantTrack",
  "trackId": "dog10k_sod1",
  "name": "Dog10K SNVs at SOD1",
  "assemblyNames": ["canFam4"],
  "adapter": {
    "type": "VcfTabixAdapter",
    "vcfGzLocation": {
      "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_sod1.vcf.gz"
    },
    "index": {
      "location": {
        "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_sod1.vcf.gz.tbi"
      }
    },
    "samplesTsvLocation": {
      "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_samples.tsv"
    }
  },
  "displays": [
    {
      "type": "LinearVariantDisplay",
      "displayId": "dog10k_sod1-LinearVariantDisplay",
      "rows": { "color": "category" }
    }
  ]
}'

In the app

In JBrowse Desktop, or in any running JBrowse Web session, open a view on this track’s assembly, then File → Open track..., choose Add track from pasted JSON, and paste:

{
  "type": "VariantTrack",
  "trackId": "dog10k_sod1",
  "name": "Dog10K SNVs at SOD1",
  "assemblyNames": ["canFam4"],
  "adapter": {
    "type": "VcfTabixAdapter",
    "vcfGzLocation": {
      "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_sod1.vcf.gz"
    },
    "index": {
      "location": {
        "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_sod1.vcf.gz.tbi"
      }
    },
    "samplesTsvLocation": {
      "uri": "https://jbrowse.org/demos/dog10k_sod1/dog10k_samples.tsv"
    }
  },
  "displays": [
    {
      "type": "LinearVariantDisplay",
      "displayId": "dog10k_sod1-LinearVariantDisplay",
      "rows": { "color": "category" }
    }
  ]
}

To put a site where the wolves have the alternate allele beside codon 40, type chr31:27122805-27122834 chr31:27123013-27123103 into the location box. The first region is 30 bp of the upstream intron, around an SNV at chr31:27,122,819.

Dog10K genotypes under SOD1, one row per canid, colored down the left edge by category. Open this view in JBrowse ↗ (large dataset — this session takes a while to load)

Opening the structure on the E40K record

A VCF record names its change on the genome's forward strand, the spelling the protein view's variant key takes. E40K's record in the slice reads chr31 27123057 . G A, so a session spec opens 7WX0 on it. Swap the gene, the PDB id and the record for your own:

{
  "views": [
    {
      "type": "ProteinView",
      "gene": "SOD1",
      "pdbId": "7WX0",
      "variant": "chr31:27123057 G>A",
      "sideBySide": true,
      "connectedView": { "loc": "chr31:27123013-27123103" }
    }
  ]
}

Try this example live ↗ ·

Open in JBrowse Desktop ↗

Needs JBrowse Desktop 5.0 or newer. If nothing opens, copy the link and paste it into Desktop's Open JBrowse Web link..., on the start screen or under File → Session.

https://jbrowse.org/code/jb2/main/?config=https%3A%2F%2Fjbrowse.org%2Fucsc%2FcanFam4%2Fconfig.json&session=spec-%7B%22views%22%3A%5B%7B%22type%22%3A%22ProteinView%22%2C%22gene%22%3A%22SOD1%22%2C%22pdbId%22%3A%227WX0%22%2C%22variant%22%3A%22chr31%3A27123057+G%3EA%22%2C%22sideBySide%22%3Atrue%2C%22connectedView%22%3A%7B%22loc%22%3A%22chr31%3A27123013-27123103%22%7D%7D%5D%7D&sessionName=JBrowse+docs+example

Open it from the link under the spec. gene finds the SOD1 transcript through the hub's search index, and connectedView.loc opens the genome view on codon 40. The launch translates the record on that transcript, labels the base with the protein change and frames residue 40 on the structure.

SOD1 at codon 40 beside 7WX0, opened on the E40K record. The band on the gene carries the change's label, and the structure labels the residue it has there.

External links

Citations

Feedback on this tutorial is welcome: contact us.

Variants on protein structures part 2: degenerative myelopathy in dogs

Prerequisites#

The data#

Opening SOD1 on codon 40 with the E40K structure#

Slicing SOD1 out of Dog10K#

E40K across breeds, village dogs and wild canids#

Opening the structure on the E40K record#

See also#

External links#

Citations#