Supplementary project data

• This webpage: https://lawlessgenomics.com/inspire2022lawless.github.io
• Manuscript and source code: https://github.com/DylanLawless/inspire2022lawless.github.io
• Pre-print on Overleaf https://www.overleaf.com/project/61718a4e077acc3d20ee68f1
• Final publication doi [citation].
• Pre-print on medarxiv: https://doi.org/10.1101/2022.06.22.22276752.
• Git commit ID and md5sum of publication version:

Study population

<iframe scrolling="no" seamless="seamless" src=" https://lawlessgenomics.com/inspire2022lawless.github.io/pages/rsv_persist2022.html " style="border:none;" height=600 width="100" ></iframe>

Figure 1: INSPIRE cohort summary. Natural quasi random RSV infection events were monitored by RT-qPCR, serology, and sequencing.

Population structure

<iframe scrolling="no" seamless="seamless" src=" https://lawlessgenomics.com/inspire2022lawless.github.io/pages/update_G_pca.html" style="border:none;" height=400 width="100"> </iframe>

To add images (B), (C) and (D).

Figure 2: Population structure. (A) Protein coding genes in RSV. (B) Phylogenetic tree based based on multiple sequence alignemnt (MSA) of amino acid G protein sequences. (C) Principal component analysis (PCA) PCs1-3 with labels indicating repeat/persistent infections from different phylogenetic clades. (D) Panel [i] summarises every pairwise genetic distance between every viral sequence. Genetic invariance in repeat/persistent infections separated by at least 15 days compared to other genetic variation within clades (panel [ii]) and within all possible pairs (panel [iii]).

Genetic association

<iframe scrolling="no" seamless="seamless" src=" https://lawlessgenomics.com/inspire2022lawless.github.io/pages/manplot.html" style="border:none;" height=300 width="60%"> </iframe>

Figure 3: Genetic association with persistent infection. (A) Amino acid association with persistent infection after multiple testing correction (significant threshold shown by dotted line).

Variant explained

This figure uses the relative MSA positions. I will update positions if we keep interative results. i.e. p.221 in relative position is p.218 in strain B reference sequence (as seen in genetic association plot signal variant).

<iframe scrolling="no" seamless="seamless" src=" https://lawlessgenomics.com/inspire2022lawless.github.io/pages/pca_variance_explaied.html" style="border:none;" height=400 width="100"> </iframe>

Figure 3 (B) Variance explained (VE) within cohort. The effect of each variant on cohort structure is shown for PCs1-2. A large % VE for a significantly associated variant would indicate a false positive. (C) Variants in strong correlation were clumped for association testing using proxies for r2 ≥ 0.8. One significant association was identified (shown in A); the r2 values for all other variants show a single highly correlated variant with the lead proxy (red).

Evidence summary

<iframe scrolling="no" seamless="seamless" src=" https://lawlessgenomics.com/inspire2022lawless.github.io/pages/gene_illustrate_rsv_Pval.html" style="border:none;" height=1000 width="100"> </iframe>

Figure 3 (D) Evidence for biological interpretation for every amino acid position is summarised.

To add

Figure 4: Supplemental: Variant clumping for reduction in association testing. [Left] Correlation between all positions. [Right] Correlation between proxy variants are clumping to remove r2 ≥ 0.8.

Figure 5: Supplemental: Publicly available RSV sequence data for > 30 years. (A) Global sample collection per year. (B) Variant associated with persistent infection tracked in public data. (C) % variance explained per year for all G protein amino acid variants from 1990-2022.

Software

• R v4.1.0 was used for data preparation and analysis http://www.r-project.org.

• R package caret was used for analysis: genetic correlations.

• R package dplyr was used for data curation.

• R package factoextra was used for analysis: PCA, and to visualise eigenvalues and variance.

• R package ggplot2 was used for data visualisation.

• R package MASS was used to analysis: logistic regression model data.

• R package stats was used for analysis: including glm for logistic regressions.

• R package stringr was used for data curation.

• R package tidyr was used for data curation.

• asn2fsa https://www.huge-man-linux.net/man1/asn2fsa.html

• clc_novo_assemble qiagenbioinformatics.com

• Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/

• GenBank https://www.ncbi.nlm.nih.gov/genbank/

• IQ-Tree https://www.iqtree.org/

• MAFFT https://mafft.cbrc.jp/alignment/software/ [citation katoh2013mafft]

• Tbl2asn https://www.ncbi.nlm.nih.gov/genbank/tbl2asn2/

• Viral Genome ORF Reader, VIGOR 3.0 https://sourceforge.net/projects/jcvi-vigor/files/

• RCSB PDB https://www.rcsb.org

• UniProt https://www.uniprot.org

Additional Data sources

• Dataset https://www.ncbi.nlm.nih.gov/bioproject/267583.
• Dataset https://www.ncbi.nlm.nih.gov/bioproject/225816.
• J. Craig Venter Institute https://www.jcvi.org.
• GenBank:NC_001989 Bovine orthopneumovirus, complete genome https://www.ncbi.nlm.nih.gov/nuccore/NC_001989.
• Reference data https://www.ncbi.nlm.nih.gov/gene/?term=1489824. G attachment glycoprotein [Human orthopneumovirus]; ID: 1489824; Location: NC_001781.1 (4675..5600); Aliases: HRSVgp07.
• Reference data https://www.ncbi.nlm.nih.gov/gene/?term=37607642. G attachment glycoprotein [Human orthopneumovirus]; ID: 37607642; Location: NC_038235.1 (4673..5595); Aliases: DZD21_gp07.
• Reference data for all public NCBI Virus https://www.ncbi.nlm.nih.gov/labs/virus/vssi/ for species: Human orthopneumovirus; genus: orthopneumovirus; family: Pneumoviridae.
• Reference data https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Nucleotide&VirusLineage_ss=Human\%20orthopneumovirus,\%20taxid:11250
• contains sequence data for Virus Lineage ss=Human orthopneumovirus, taxid:11250 nucleotide: 26’965, protein: 53’804, RefSeq Genomes: 2.
• Reference https://www.ncbi.nlm.nih.gov/protein/NP_056862.1
• GCF_002815475.1 (release 2018-08-19) Nucleotide Accessions: NC_038235.1, protein: Y_009518856.1
• Reference https://www.ncbi.nlm.nih.gov/protein/YP_009518856.1
• GCF_000855545.1 (release 2015-02-12) Nucleotide Accessions: NC_001781.1, protein: NP_056862.1 (strain B1).

Repository maintenance

Using a custom host and ssh key is recommended for maintenance.

## Set up the ssh config file
cd ~/.ssh/config

## Set such that Host and User are custom
# lawlessgenomics repo
Host dylanlawless.github.com
  HostName github.com
  User DylanLawless
  PreferredAuthentications publickey
  IdentityFile ~/.ssh/key1_rsa
  IdentitiesOnly yes


# Clone using the correct Host as per config.
git clone git@dylanlawless.github.com:DylanLawless/inspire2022lawless.github.io.git

# Set the local user here (instead of global, i.e. /Users/user/.gitconfig)
cd "the clone repo dir"
git config user.email personemail@addess.com
git config user.name DylanLawless

Colophon

This page uses the body font Switzer-Light, a neo-grotesk Latin-script typeface with 18 styles, designed by Jérémie Hornus. Body text color is #0a2e4a and additional colors are found in _sass/variables.scss The layout is based on the jekyll-theme-cayman. It was developed using a local installation of the Jekyll Ruby gem and published using GitHub Pages. Add the methods section for all data access and software here.